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Chapter 36 Glial cell development and function in the rat optic nerve
F. J. Seil, E. Herbert and B. M. Carlson (Eds.) Progress in Brain Research, Vol. 71
0 1987 Elsevier Science Publishers B.V.,
Biomedical Division
435
CHAPTER 36
Glial cell development and function in the rat optic nerve Martin C. Raff, Sally Temple and Charles ffrench-Constant Medical Research Council Developmental Neurobiology Project, Zoology Department, University College London, London WCIE 6BT. England
Introduction Glial cells are thought to play an important part in neural development and repair but how they do so is uncertain. We have studied the development and properties of glial cells in the rat optic nerve, one of the simplest parts of the central nervous system (CNS). Some of these findings may prove to be helpful in understanding the role of glial cells in CNS regeneration. Three types of macroglial cells in optic nerve The rat optic nerve contains three types of macroglial cells, oligodendrocytes and two types of astrocytes. Type-1 astrocytes form the glial limiting membrane at the periphery of the nerve and type2 astrocytes occupy the interior of the nerve and correspond to ‘fibrous’ or ‘fibrillary’ astrocytes (Miller and Raff, 1984). In vitro studies suggest that the three types of macroglial cells arise by two distinct lineages: oligodendrocytes and type-2 astrocytes develop from a common, bipotential (0-2A) progenitor cell (Raff et al., 1983a), whereas type-1 astrocytes develop from a different precursor cell (Raff et al., 1984). Type-1 astrocytes first appear at embryonic day 16 (E16), oligodendrocytes on the day of birth (E21), and type-2 astrocytes between postnatal days 8 and 10 (P8-10) (Miller et al., 1985). Controls of glial cell differentiation What determines the choice of developmental path-
way taken by the 0-2A progenitor cell and what controls the timing of its differentiation? Cell culture studies have provided some tentative answers. When perinatal optic nerve cells are cultured, the 0-2A progenitor cells stop dividing and differentiate within 2-3 days. In 1&20% fetal calf serum (FCS), most develop into type-2 astrocytes, while in 0-1 YO FCS most develop into oligodendrocytes (Raff et al., 1983a). This differentiation in vitro is premature; in vivo oligodendrocytes (Skoff et al., 1976a,b) and then type-2 astrocytes (Miller et al., 1985) are produced from dividing progenitor cells over a period of many weeks. However, the normal timing of oligodendrocytedevelopment seen in vivo can be reconstituted in vitro by the addition of type-1 astrocytes to the culture. When El7 optic nerve cells are grown in < 1YOFCS on a monolayer of type-1 astrocytes or in medium conditioned by type-1 astrocytes, oligodendrocytes first appear after 4 days, equivalent to the time they first appear in vivo, and new oligodendrocytes continue to be produced from dividing progenitor cells for several weeks (Raff et al., 1985). On the basis of these and other findings, we hypothesized (Raff et al., 1985) that the timing of oligodendrocyte differentiation may be controlled in the following way: type- 1 astrocytes, the first glial cells to differentiate in the optic nerve, secrete growth factors that stimulate 0-2A progenitor cells to proliferate (Noble and Murray, 1984; Raff et al., 1985). The progenitor cell itself determines when it differentiates into an oligodendrocyte by counting the number of times it divides; after a certain number of cell divisions it becomes unresponsive to the growth factors, stops dividing and
436
constitutively differentiates into an oligodendrocyte. The first 0-2A progenitor cells reach this point around birth, while others reach it at various times during the first weeks after birth. Progenitor cells that are cultured in < 1YOFCS with an inadequate concentration of growth factors prematurely stop dividing and differentiate into oligodendrocytes. The results of single cell experiments are consistent with this hypothesis. When individual 0-2A progenitor cells are cultured on their own in microwells in < 1% FCS, they drop out of division and develop into oligodendrocytes (Temple and Raff, 1985), indicating that signals from other cells are not required to induce this differentiation. On the other hand, when single 0-2A progenitor cells are cultured in microwells on a monolayer of type-l astrocytes, they undergo a period of proliferation, following which the daughter cells differentiate into oligodendrocytes more or less together. While the number of divisions preceding differentiation is largely the same for the cells of a single clone, as would be expected if the timing of differentiation were controlled by counting divisions, the number of times individual progenitor cells in different microwells divide before differentiation in these experiments varies between one and eight (Temple and Raff, 1986). Why are the progenitor cells heterogeneous in their proliferative capacity? An attractive possibility is that they are continually produced from pre-progenitor cells over a prolonged period, so that the number of divisions the progenitor cells will have gone through before being cultured is variable. The finding of proliferating 0-2A progenitor cells in adult rat optic nerve (ffrenchConstant and Raff, 1986a) is consistent with the possibility that, since the maximum number of divisions 0-2A progenitor cells go through in vitro is small, the presence of such cells in adult nerve suggests that they may be produced continually throughout life from slowly dividing, self renewing stem cells. The timing of type-2 astrocyte differentiation seems to be controlled by a different mechanism from that controlling oligodendrocyte differentiation. When El7 optic nerve cells are cultured on
type-1 astrocyte monolayers in 10% FCS, 0-2A progenitor cells prematurely stop dividing and differentiate into type-2 astrocytes within 3 days, at least a week before they first appear in vivo (Raff et al., 1985). On the other hand, when they are cultured in the same way, but in < l % FCS, type-2 astrocytesdo not develop in vitro, even after several weeks (Raff et al., 1985). These results suggest that type-2 astrocyte differentiation requires an inducer, which we can mimic with 10% FCS in vitro but which does not appear in the developing optic nerve until the second postnatal week. The nature and source of this putative inducer are unknown.
Glial cell functions While oligodendrocytes form myelin sheaths around axons, thereby increasing the velocity and efficiency of action potential propagation, the functions of astrocytes are still uncertain. However, the finding that the two types of astrocytes in optic nerve are biochemically and developmentally distinct makes it likely that they have different functions. One of the only established functions of astrocytes is to form scar tissue in response to injury in the CNS, a process called reactive gliosis. We have found that the astrocytes forming the glial scar in adult rat optic nerve 20 weeks after nerve transection have the antigenic phenotype of type-1 astrocytes (Miller et al., 1986). Although we cannot exclude that type-2 astrocytes (which comprise 65% of the astrocytes in adult optic nerve) change their antigenic phenotype in response to nerve transection and come to resemble type-I astrocytes, quantitative immunohistochemical analyses of cut nerves suggest that type-2 astrocytes (and oligodendrocytes) eventually die in transected nerves and that type-I astrocytes are mainly responsible for the gliosis (Miller et al., 1986). The same results are obtained following stab lesions in the corpus callosum (Miller et al., 1986). Similarly, very few 0-2A lineage cells are found in optic nerves examined 2 and 8 weeks after neonatal transection; most of the cells appear to be type-I astrocytes (David et al., 1984). Taken together, these results suggest that
431
0-2A lineage cells depend on axons for their long term survival and that gliotic scar formation following trauma or Wallerian degeneration in white matter is largely a function of type-1 astrocytes. What then is the function of type-2 astrocytes in optic nerve? Since they seem to occur primarily in white matter, they may have specific functions related to myelinated axons. For example, astrocyte processes surround nodes of Ranvier in white matter where, in principle, they could help to stabilize local extracellular ion concentrations in the face of repeated nerve impulses. But do these perinodal astrocyte processes come from type-2 astrocytes? We have recently obtained indirect evidence that most, if not all, of them do. Initially, we found that the monoclonal antibody HNK- 1, which recognizes an oligosaccharide shared by a number of cell adhesion molecules (Kruse et al., 1984), preferentially labels perinodal astrocyte processes in adult rat optic nerve (ffrench-Constant et al., 1986a). We then showed that the major HNK-1 positive molecule concentrated on perinodal astrocyte processes in the optic nerve is the J1 glycoprotein (ffrench-Constant et al., 1986a), a cell adhesion molecule previously shown to mediate neuron-astrocyte adhesion in vitro (Kruse et al., 1985). Finally, we found that in cultures of perinatal optic nerve J1 is detected only on 0-2A lineage cells, including type-2 astrocytes, but not on type-1 astrocytes (ffrenchConstant and Raff, 1986b).
atively late in ontogeny (since myelination occurs late) and that they die in the absence of axons (without axons to serve, there is no reason for their continued survival). It also makes sense that oligodendrocytes should be produced by a constitutive process before type-2 astrocytes so that all appropriate axons are ensheathed before 0-2A progenitor cells are induced to develop into type-2 astrocytes, which then extend processes around developing nodes of Ranvier. While most of our studies have been directed at understanding how glial cells develop in the optic nerve, two of our findings have a direct bearing on regeneration in the mammalian CNS. The first is that glial scar formation following injury to white matter seems to be largely a property of a relatively minor subpopulation of astrocytes (Miller et al., 1986). The second is that proliferating 0-2A progenitor cells are present in the adult CNS (ffrench-Constant and Raff, 1986a), raising the possibility that they may be able to produce new oligodendrocytes and type-2 astrocytes following injury and thereby aid regeneration.
Conclusion
References
Our initial studies on the optic nerve produced two surprises: first, that there are two biochemically distinct types of astrocytes in the nerve (Raff et al., 1983b; Miller and Raff, 1984) and second, that oligodendrocytes and type-2 astrocytes (but not type-1 astrocytes) develop from a common progenitor cell (Raff et al., 1983a), a lineage quite different from that described in textbooks. In light of our subsequent studies, these findings make teleological sense. If the oligodendrocyte-type-2 astrocyte lineage is viewed as specialized for myelination, then it is not surprising that these cells differentiate rel-
David, S.,Miller, R. H., Patel, R. and Raff, M. C. (1984) Effects of neonatal transection on glial cell development in the rat optic nerve: evidence that the oligodendrocyte-type-2 astrocyte cell lineage depends on axons for its survival. J. Neuro~ y t o l .13: . 961-974. ffrench-Constant, C. and Raff, M. C. (1986a) Proliferating bipotential glial progenitor cells in adult rat optic nerve. Nature, 319: 499-502. ffrench-Constant, C. and Raff, M. C. (1986b) The oligodendrocyt&ype-2 astrocyte cell lineage is specialized for myelination. Nature 323: 335-338. ffrench-Constant, C., Miller, R. H., Kruse, J., Schachner, M. and Raff, M. C. (1986) Molecular specialization of astrocyte processes at nodes of Ranvier in rat optic nerve. J. Cell, Biol., 102 844852.
Acknowledgements
Sally Temple was supported by a Medical Research Council studentship and Charles ffrench-Constant by a grant from the British Multiple Sclerosis Society.
438 Kruse, J., Mailhammer, R., Wernecke, H., Faissner, A., Sommer, I., Goridis, C. and Schachner, M. (1984) Neural cell adhesion molecules and myelin associated glywprotein share a common carbohydrate moiety recognised by monoclonal antibodies L2 and HNK-I. Nature, 311: 153-155. Kruse, J., Keilhauer, G., Faissner, A., Tmpl, R.and Schachner, M. (1985) The J1 glycoprotein -a novel nervous system cell adhesion molecule of the L2/HNK-I family. Nature, 316: 146-148. Miller, R. H. and RaK,M. C. (1984) Fibrous and protoplasmic astrocytes are biochemically and developmentallydistinct. J. Neurosci.. 4 585-592. Miller, R. H., David, S., Patel, R.,Abney, E. R. and M, M. C. (1985) A quantitative imniunohistochemical study of macroghal cell development in the rat optic nerve: In vivo evidence for two distinct astrocyte lineages. Dev. Biol., 111: 35-41. Miller, R. H., Abney, E. R., David, S., ffrench-Constant, C., Lindsay, R., Patel, R.,Stone, J. and R&, M.C. (1986) Is reactive gliosis a property of a distinct subpopulation of astrocytes? J. Neurosci., 6: 22-29. Noble, M. and Murray, K. (1984) Purified astrocytes promote the in vitro division of a bipotential glial progenitor cell. EMBO J., 3: 2243-2247. Raff, M. C., Miller, R. H. and Noble, M.(1983a) A glial progenitor cell that develops in vitro into an astrocyte or an oli-
godendrocyte depending on the culture medium. Nature, 303: 390-396. RafT, M.C., Abney, E. R., Cohen, J., Lindsay, R. and Noble, M. (198313) Two types of astrocytes in culture of developing rat white matter: differences in morphology, surface gangliosides and growth characteristics. J . Neurosci., 3: 1289-1300. R&, M.C., Abney, E. R. and Miller, R. H. (1984) Two glial cell lineages diverge prenatally in rat optic nerve. Dev. Biol., 106: 53-60. RalT, M. C., Abney, E. R. and Fok-Seang, J. (1985) Reconstitution of a developmental clock in vitro: A critical role for astrocytes in the timing of oligodendrocyte differentiation. Cell, 4 2 61-69. Skoff, R., Price, D. and Stocks, A. (1976a) Electron microscopic autoradiographic studies of gliogenesis in rat optic nerve. I. Cell proliferation. J. Comp. Neurol.. 169 291-312. Skoff,R., Price,D. and Stocks, A. (1976b) Electron microscopic autoradiographic studies of gliogenesis in rat optic nerve. 11. Time of origin. J. Comp. Neurol., 169: 313-333. Temple, S. and RaK, M. C. (1985) Differentiation of a bipotential dial progenitor cell in single cell microculture. Nature, 313: 223-225. Temple, S. and RaK, M. C. (1986) Clonal analysis of oligodendrocyte development in culture: evidence for a developmental clock that wunts cell divisions. Cell, 44: 773-779.
0 1987 Elsevier Science Publishers B.V.,
Biomedical Division
435
CHAPTER 36
Glial cell development and function in the rat optic nerve Martin C. Raff, Sally Temple and Charles ffrench-Constant Medical Research Council Developmental Neurobiology Project, Zoology Department, University College London, London WCIE 6BT. England
Introduction Glial cells are thought to play an important part in neural development and repair but how they do so is uncertain. We have studied the development and properties of glial cells in the rat optic nerve, one of the simplest parts of the central nervous system (CNS). Some of these findings may prove to be helpful in understanding the role of glial cells in CNS regeneration. Three types of macroglial cells in optic nerve The rat optic nerve contains three types of macroglial cells, oligodendrocytes and two types of astrocytes. Type-1 astrocytes form the glial limiting membrane at the periphery of the nerve and type2 astrocytes occupy the interior of the nerve and correspond to ‘fibrous’ or ‘fibrillary’ astrocytes (Miller and Raff, 1984). In vitro studies suggest that the three types of macroglial cells arise by two distinct lineages: oligodendrocytes and type-2 astrocytes develop from a common, bipotential (0-2A) progenitor cell (Raff et al., 1983a), whereas type-1 astrocytes develop from a different precursor cell (Raff et al., 1984). Type-1 astrocytes first appear at embryonic day 16 (E16), oligodendrocytes on the day of birth (E21), and type-2 astrocytes between postnatal days 8 and 10 (P8-10) (Miller et al., 1985). Controls of glial cell differentiation What determines the choice of developmental path-
way taken by the 0-2A progenitor cell and what controls the timing of its differentiation? Cell culture studies have provided some tentative answers. When perinatal optic nerve cells are cultured, the 0-2A progenitor cells stop dividing and differentiate within 2-3 days. In 1&20% fetal calf serum (FCS), most develop into type-2 astrocytes, while in 0-1 YO FCS most develop into oligodendrocytes (Raff et al., 1983a). This differentiation in vitro is premature; in vivo oligodendrocytes (Skoff et al., 1976a,b) and then type-2 astrocytes (Miller et al., 1985) are produced from dividing progenitor cells over a period of many weeks. However, the normal timing of oligodendrocytedevelopment seen in vivo can be reconstituted in vitro by the addition of type-1 astrocytes to the culture. When El7 optic nerve cells are grown in < 1YOFCS on a monolayer of type-1 astrocytes or in medium conditioned by type-1 astrocytes, oligodendrocytes first appear after 4 days, equivalent to the time they first appear in vivo, and new oligodendrocytes continue to be produced from dividing progenitor cells for several weeks (Raff et al., 1985). On the basis of these and other findings, we hypothesized (Raff et al., 1985) that the timing of oligodendrocyte differentiation may be controlled in the following way: type- 1 astrocytes, the first glial cells to differentiate in the optic nerve, secrete growth factors that stimulate 0-2A progenitor cells to proliferate (Noble and Murray, 1984; Raff et al., 1985). The progenitor cell itself determines when it differentiates into an oligodendrocyte by counting the number of times it divides; after a certain number of cell divisions it becomes unresponsive to the growth factors, stops dividing and
436
constitutively differentiates into an oligodendrocyte. The first 0-2A progenitor cells reach this point around birth, while others reach it at various times during the first weeks after birth. Progenitor cells that are cultured in < 1YOFCS with an inadequate concentration of growth factors prematurely stop dividing and differentiate into oligodendrocytes. The results of single cell experiments are consistent with this hypothesis. When individual 0-2A progenitor cells are cultured on their own in microwells in < 1% FCS, they drop out of division and develop into oligodendrocytes (Temple and Raff, 1985), indicating that signals from other cells are not required to induce this differentiation. On the other hand, when single 0-2A progenitor cells are cultured in microwells on a monolayer of type-l astrocytes, they undergo a period of proliferation, following which the daughter cells differentiate into oligodendrocytes more or less together. While the number of divisions preceding differentiation is largely the same for the cells of a single clone, as would be expected if the timing of differentiation were controlled by counting divisions, the number of times individual progenitor cells in different microwells divide before differentiation in these experiments varies between one and eight (Temple and Raff, 1986). Why are the progenitor cells heterogeneous in their proliferative capacity? An attractive possibility is that they are continually produced from pre-progenitor cells over a prolonged period, so that the number of divisions the progenitor cells will have gone through before being cultured is variable. The finding of proliferating 0-2A progenitor cells in adult rat optic nerve (ffrenchConstant and Raff, 1986a) is consistent with the possibility that, since the maximum number of divisions 0-2A progenitor cells go through in vitro is small, the presence of such cells in adult nerve suggests that they may be produced continually throughout life from slowly dividing, self renewing stem cells. The timing of type-2 astrocyte differentiation seems to be controlled by a different mechanism from that controlling oligodendrocyte differentiation. When El7 optic nerve cells are cultured on
type-1 astrocyte monolayers in 10% FCS, 0-2A progenitor cells prematurely stop dividing and differentiate into type-2 astrocytes within 3 days, at least a week before they first appear in vivo (Raff et al., 1985). On the other hand, when they are cultured in the same way, but in < l % FCS, type-2 astrocytesdo not develop in vitro, even after several weeks (Raff et al., 1985). These results suggest that type-2 astrocyte differentiation requires an inducer, which we can mimic with 10% FCS in vitro but which does not appear in the developing optic nerve until the second postnatal week. The nature and source of this putative inducer are unknown.
Glial cell functions While oligodendrocytes form myelin sheaths around axons, thereby increasing the velocity and efficiency of action potential propagation, the functions of astrocytes are still uncertain. However, the finding that the two types of astrocytes in optic nerve are biochemically and developmentally distinct makes it likely that they have different functions. One of the only established functions of astrocytes is to form scar tissue in response to injury in the CNS, a process called reactive gliosis. We have found that the astrocytes forming the glial scar in adult rat optic nerve 20 weeks after nerve transection have the antigenic phenotype of type-1 astrocytes (Miller et al., 1986). Although we cannot exclude that type-2 astrocytes (which comprise 65% of the astrocytes in adult optic nerve) change their antigenic phenotype in response to nerve transection and come to resemble type-I astrocytes, quantitative immunohistochemical analyses of cut nerves suggest that type-2 astrocytes (and oligodendrocytes) eventually die in transected nerves and that type-I astrocytes are mainly responsible for the gliosis (Miller et al., 1986). The same results are obtained following stab lesions in the corpus callosum (Miller et al., 1986). Similarly, very few 0-2A lineage cells are found in optic nerves examined 2 and 8 weeks after neonatal transection; most of the cells appear to be type-I astrocytes (David et al., 1984). Taken together, these results suggest that
431
0-2A lineage cells depend on axons for their long term survival and that gliotic scar formation following trauma or Wallerian degeneration in white matter is largely a function of type-1 astrocytes. What then is the function of type-2 astrocytes in optic nerve? Since they seem to occur primarily in white matter, they may have specific functions related to myelinated axons. For example, astrocyte processes surround nodes of Ranvier in white matter where, in principle, they could help to stabilize local extracellular ion concentrations in the face of repeated nerve impulses. But do these perinodal astrocyte processes come from type-2 astrocytes? We have recently obtained indirect evidence that most, if not all, of them do. Initially, we found that the monoclonal antibody HNK- 1, which recognizes an oligosaccharide shared by a number of cell adhesion molecules (Kruse et al., 1984), preferentially labels perinodal astrocyte processes in adult rat optic nerve (ffrench-Constant et al., 1986a). We then showed that the major HNK-1 positive molecule concentrated on perinodal astrocyte processes in the optic nerve is the J1 glycoprotein (ffrench-Constant et al., 1986a), a cell adhesion molecule previously shown to mediate neuron-astrocyte adhesion in vitro (Kruse et al., 1985). Finally, we found that in cultures of perinatal optic nerve J1 is detected only on 0-2A lineage cells, including type-2 astrocytes, but not on type-1 astrocytes (ffrenchConstant and Raff, 1986b).
atively late in ontogeny (since myelination occurs late) and that they die in the absence of axons (without axons to serve, there is no reason for their continued survival). It also makes sense that oligodendrocytes should be produced by a constitutive process before type-2 astrocytes so that all appropriate axons are ensheathed before 0-2A progenitor cells are induced to develop into type-2 astrocytes, which then extend processes around developing nodes of Ranvier. While most of our studies have been directed at understanding how glial cells develop in the optic nerve, two of our findings have a direct bearing on regeneration in the mammalian CNS. The first is that glial scar formation following injury to white matter seems to be largely a property of a relatively minor subpopulation of astrocytes (Miller et al., 1986). The second is that proliferating 0-2A progenitor cells are present in the adult CNS (ffrench-Constant and Raff, 1986a), raising the possibility that they may be able to produce new oligodendrocytes and type-2 astrocytes following injury and thereby aid regeneration.
Conclusion
References
Our initial studies on the optic nerve produced two surprises: first, that there are two biochemically distinct types of astrocytes in the nerve (Raff et al., 1983b; Miller and Raff, 1984) and second, that oligodendrocytes and type-2 astrocytes (but not type-1 astrocytes) develop from a common progenitor cell (Raff et al., 1983a), a lineage quite different from that described in textbooks. In light of our subsequent studies, these findings make teleological sense. If the oligodendrocyte-type-2 astrocyte lineage is viewed as specialized for myelination, then it is not surprising that these cells differentiate rel-
David, S.,Miller, R. H., Patel, R. and Raff, M. C. (1984) Effects of neonatal transection on glial cell development in the rat optic nerve: evidence that the oligodendrocyte-type-2 astrocyte cell lineage depends on axons for its survival. J. Neuro~ y t o l .13: . 961-974. ffrench-Constant, C. and Raff, M. C. (1986a) Proliferating bipotential glial progenitor cells in adult rat optic nerve. Nature, 319: 499-502. ffrench-Constant, C. and Raff, M. C. (1986b) The oligodendrocyt&ype-2 astrocyte cell lineage is specialized for myelination. Nature 323: 335-338. ffrench-Constant, C., Miller, R. H., Kruse, J., Schachner, M. and Raff, M. C. (1986) Molecular specialization of astrocyte processes at nodes of Ranvier in rat optic nerve. J. Cell, Biol., 102 844852.
Acknowledgements
Sally Temple was supported by a Medical Research Council studentship and Charles ffrench-Constant by a grant from the British Multiple Sclerosis Society.
438 Kruse, J., Mailhammer, R., Wernecke, H., Faissner, A., Sommer, I., Goridis, C. and Schachner, M. (1984) Neural cell adhesion molecules and myelin associated glywprotein share a common carbohydrate moiety recognised by monoclonal antibodies L2 and HNK-I. Nature, 311: 153-155. Kruse, J., Keilhauer, G., Faissner, A., Tmpl, R.and Schachner, M. (1985) The J1 glycoprotein -a novel nervous system cell adhesion molecule of the L2/HNK-I family. Nature, 316: 146-148. Miller, R. H. and RaK,M. C. (1984) Fibrous and protoplasmic astrocytes are biochemically and developmentallydistinct. J. Neurosci.. 4 585-592. Miller, R. H., David, S., Patel, R.,Abney, E. R. and M, M. C. (1985) A quantitative imniunohistochemical study of macroghal cell development in the rat optic nerve: In vivo evidence for two distinct astrocyte lineages. Dev. Biol., 111: 35-41. Miller, R. H., Abney, E. R., David, S., ffrench-Constant, C., Lindsay, R., Patel, R.,Stone, J. and R&, M.C. (1986) Is reactive gliosis a property of a distinct subpopulation of astrocytes? J. Neurosci., 6: 22-29. Noble, M. and Murray, K. (1984) Purified astrocytes promote the in vitro division of a bipotential glial progenitor cell. EMBO J., 3: 2243-2247. Raff, M. C., Miller, R. H. and Noble, M.(1983a) A glial progenitor cell that develops in vitro into an astrocyte or an oli-
godendrocyte depending on the culture medium. Nature, 303: 390-396. RafT, M.C., Abney, E. R., Cohen, J., Lindsay, R. and Noble, M. (198313) Two types of astrocytes in culture of developing rat white matter: differences in morphology, surface gangliosides and growth characteristics. J . Neurosci., 3: 1289-1300. R&, M.C., Abney, E. R. and Miller, R. H. (1984) Two glial cell lineages diverge prenatally in rat optic nerve. Dev. Biol., 106: 53-60. RalT, M. C., Abney, E. R. and Fok-Seang, J. (1985) Reconstitution of a developmental clock in vitro: A critical role for astrocytes in the timing of oligodendrocyte differentiation. Cell, 4 2 61-69. Skoff, R., Price, D. and Stocks, A. (1976a) Electron microscopic autoradiographic studies of gliogenesis in rat optic nerve. I. Cell proliferation. J. Comp. Neurol.. 169 291-312. Skoff,R., Price,D. and Stocks, A. (1976b) Electron microscopic autoradiographic studies of gliogenesis in rat optic nerve. 11. Time of origin. J. Comp. Neurol., 169: 313-333. Temple, S. and RaK, M. C. (1985) Differentiation of a bipotential dial progenitor cell in single cell microculture. Nature, 313: 223-225. Temple, S. and RaK, M. C. (1986) Clonal analysis of oligodendrocyte development in culture: evidence for a developmental clock that wunts cell divisions. Cell, 44: 773-779.