Glial Cell Lineages in the Rat Cerebral Cortex

Experimental Neurology 156, 418–429 (1999) Article ID exnr.1999.7044, available online at http://www.idealibrary.com on

Glial Cell Lineages in the Rat Cerebral Cortex John G. Parnavelas Department of Anatomy and Developmental Biology, University College London, London WC1E 6BT, United Kingdom Received December 14, 1998; accepted January 18, 1999

I have traced the fates of glial cell progenitors in the rat cerebral cortex marked with a recombinant retrovirus throughout most of the period of corticogenesis, from embryonic (E) day 14 to postnatal (P) day 14. Discrete clusters of clonally related glia were examined in serially cut sections, and their phenotypes identified using reliable light and electron microscopic criteria. Analysis of a large number of clones marked with retrovirus at various stages of embryonic life contained, with very few exceptions, either all astrocytes or all oligodendrocytes. This observation suggests that the ventricular zone contains separate progenitor cells for the two glial cell types. Oligodendrocyte clones were rarely seen in the cortices injected with retrovirus at the early stages of corticogenesis (E14–E16), suggesting that there is a very small number of oligodendrocyte progenitors in the ventricular zone at these early stages. Their frequency increased significantly at later embryonic ages. At these later stages, ventricular zone cells also give rise to progenitor cells that make up the subventricular zone in early postnatal life. Injections of retrovirus in this proliferative zone shortly after birth resulted in the generation of labeled astrocyte and oligodendrocyte clones in the cortical gray and white matter, with the astrocyte clones being in the majority. Injections at increasingly later stages resulted in the presence, predominantly in the white matter of both hemispheres and in the corpus callosum, of progressively more oligodendrocyte clones and fewer astrocyte clones. Injections at P14 generated only oligodendrocyte clones in the white matter of both hemispheres. A small number of clusters (F10%) generated after subventricular zone injections contained both astrocytes and oligodendrocytes, suggesting that single subventricular zone cells can differentiate into both glial cell types. r 1999 Academic Press Key Words: glia; lineage; cerebral cortex; retrovirus; astrocytes; oligodendrocytes.

INTRODUCTION

The existence of glial cells has been known from about the middle of the 19th century and, although 0014-4886/99 $30.00 Copyright r 1999 by Academic Press All rights of reproduction in any form reserved.

there were antecedent publications, their discovery is generally attributed to Virchow, who coined the term neuroglia in 1859 (see Ref. 28 for historical review). The early observations clearly allowed the heterogeneous family of neuroglia to be subdivided into astrocytes and oligodendrocytes (together comprising the macroglia), microglia, and ependyma. The progenitors of glial cells were then thought to be the epithelial cells of the neural tube, which were called ‘‘spongioblasts’’ by His in 1887 (see Ref. 7 for review). These spongioblasts were considered to be the progenitors of all the nonneural cells which were derived from the neuroectoderm. Later research confirmed the ectodermal origin of the astrocytes and oligodendrocytes, but the microglia, like the macrophages of other tissues, appear to be of mesodermal origin. His’ recognition of the presence of glial progenitors within the ventricular zone was corroborated nearly 100 years later by immunocytochemical studies at the light and electron microscopical levels in primates and humans (2, 11). Results from a number of studies, which employed morphological, immunohistochemical, and thymidine labeling techniques, have suggested that the two germinal zones in the developing telencephalon, the ventricular (VZ) and subventricular (SVZ) zones, are the sources of the neurons and glia of the cerebral cortex. The VZ gives origin to both neurons and glia and disappears at the early stages of development, while the SVZ contains proliferating cells that mainly give rise to macroglia (7). This zone expands greatly in late gestation, and in early postnatal life it comes to reside adjacent to the lateral ventricles and just underneath the formative white matter (5, 32). Although there has been considerable interest in gliogenesis over the years (see Refs. 1, 28, and 30 for reviews), information on glial cell development is still deficient in many respects. The question of the ontogeny of glia has been bedevilled by the fact that there is long delay between the time of origin of cells and the time at which they reach their final state of differentiation. During this period they normally undergo many divisions both in the germinal zones and in the extraventricular sites. Because of successive mitoses, attempts

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to use thymidine autoradiography to determine the time of origin of glia are hampered by the dilution of the label in these cells below detectable levels. Other studies, which relied on morphological criteria to study the developmental fates of immature glial cells, proved largely unsuccessful because it was not possible to assign immature cells to specific lineages. More recent efforts used cell-specific antibodies to overcome the problem of cell identification, but it remained difficult to determine lineage relationships between the various cell types (8, 11). However, the method of retrovirusmediated gene transfer, when combined with reliable morphological criteria and/or cell-specific markers, allows assessment of the degree of phenotype homogeneity within individual clones and reliable identification of the nature of the progeny of different progenitors. Retrovirus-mediated gene transfer has been applied to the question of glial cell lineage in the cerebral cortex. Studies involved either the use of primary cultures of developing rodent neocortex (15, 33, 35) or injections of retrovirus in developing animals. In the latter group of studies, recombinant retrovirus was injected in very few stages of a rather extensive period of gliogenesis that spans several weeks in rats. Specifically, some studies (16, 25) examined clusters of clonally related cells in rats that had previously received injections in the lateral ventricles at embryonic days (E) 15–16. These clusters, in agreement with previous work in the mouse (15), for the most part contained cells of glial or neuronal phenotype, suggesting that separate progenitor cells for each of these phenotypes exist in the VZ at that stage of corticogenesis. However, it is not known whether progenitor cells committed to specific glia lineages are present in the VZ before E15–E16 or during the later stages of embryonic life when the VZ diminishes in prominence and the SVZ becomes the main germinal zone of the developing cortex. Other studies examined the fates of glial progenitors in the cerebral cortex following injections of recombinant retrovirus into the SVZ either shortly after birth (9, 10, 14) or at 2 weeks of age (10). The results of these studies have suggested that the course of development and disposition of the progeny of SVZ cells differ according to the stage of cortical gliogenesis. Here, I investigated the fates of glial progenitors in the rat cerebral cortex marked with recombinant retrovirus throughout most of the period of corticogenesis. Specifically, such cells were infected with retrovirus at every day during the last week of gestation (E14–E20), which coincides with the period of cortical neurogenesis, and at a number of postnatal ages during the first 2 weeks of life when the great majority of glial cells are generated (17).

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MATERIALS AND METHODS

Prenatal Retrovirus Injections Female Sprague–Dawley albino rats were bred to obtain fetuses of known gestational ages. They were mated at night, and the first day of pregnancy, assessed by the presence of a vaginal plug, was designated E1. Four to ten pregnant animals at each day of gestation between E14 and E20 were used in this part of the study. They were anesthetized with halothane, and after midline laparotomy, fetal heads were transilluminated with a fiber optics source, and the location of the lateral ventricles was identified. The ␺ BAG retrovirus, which carries the lacZ reporter gene (16), was injected into the ventricles of each embryo. Approximately 1 µl of retroviral suspension, at a dilution of 105–106 colonyforming units/ml, containing Polybrene (0.005%) and fast green (0.01%), was injected through a 33-gauge needle. After all the fetuses were injected, the abdominal wall was sutured and the pregnant animals were allowed to recover in individual cages. Postnatal Retrovirus Injections Ten Sprague–Dawley rats at each of the ages 2, 7, 9, 11, and 14 days after birth were anesthetized with Hypnon. They were then positioned in a stereotaxic apparatus, the scalp was deflected, and approximately 1 µl of BAG retroviral stock was injected unilaterally, through a 27-gauge needle attached to a Hamilton microsyringe, in the area adjacent to the dorsolateral portion of the lateral ventricle. This is the region in which most cells of the persisting SVZ are located in postnatal life (5). Following the injections, the pups were revived by warming before they were returned to their mothers. Histochemistry and Tissue Processing The injected fetuses were born normally. All pre- and postnatally injected rats were allowed to survive for 6–9 weeks, except for two litters of rats injected at E19 and sacrificed at 7 days after birth, and six rats that were sacrificed 3 days after receiving retrovirus injections in the SVZ at 9 days of age. All rats were deeply anesthetized with ether. The older animals were perfused through a cannula tied into the ascending aorta with a fixative solution containing 4% paraformaldehyde and 0.2% glutaraldehyde in 0.2 M phosphate buffer (PB; pH 7.4). The young rats were perfused with the same fixative solution delivered through a hypodermic needle in the left cardiac ventricle. Following the perfusion, the brains were removed from the skulls and stored in fixative without glutaraldehyde for about 30 min. The cerebral hemispheres were separated in all cases, except for the animals injected postnatally in which the hemispheres

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FIG. 1. Examples of the light microscopic appearance of ␤-gal⫹ glial cells in the adult cerebral cortex (A, B) and in the SVZ (C) following intraventricular injections of retrovirus. (A) Astrocytes showing the characteristic veil-like processes and end feet (arrow) on blood vessels (BV). These cells are part of the clone illustrated in Fig. 4A. (B) A tightly packed cluster of oligodendrocytes in the subcortical white matter showing the characteristic parallel processes. (C) Cells in the SVZ in the brain of a P7 rat resulting from an injection of retrovirus at E19. These cells have fairly thick processes pointing in the direction of the lateral ventricle (LV). ⫻750.

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FIG. 2. Electron micrographs of representative ␤-gal⫹ astrocytes in the cortical gray matter. The reaction product has a punctate appearance and is distributed predominantly around the nucleus. As is typical of astrocytes, these cells show electron-lucent cytoplasm that includes bundles of glial filaments (arrows). Cell shown in A is part of the clone illustrated in Fig. 4A, and that shown in B is part of the clone illustrated in Fig. 4C. ⫻8000.

were kept together, and sectioned serially in the coronal plane at 100 µm using a Vibratome. The sections were collected in PB and then stained histochemically for ␤-galactosidase (␤-gal⫹), using X-Gal as a substrate. All sections from hemispheres that showed under the dissecting microscope to contain ␤-gal⫹ cells were subsequently processed for electron microscopy. Briefly, they were rinsed twice in PB, postfixed for 1 h in 1% OsO4, rinsed in 0.1 M acetate buffer, stained for 1 h in 1% aqueous uranyl acetate, rinsed in acetate buffer again, dehydrated through an ethanol series, and flatembedded serially on microscope slides. Analysis of Material All sections were examined with the light microscope and every ␤-gal⫹ cell was mapped in camera lucida drawings of the serially arranged sections. In accordance with earlier lineage studies in the cortex (16, 20,

25), discrete clusters of closely spaced ␤-gal⫹ cells, separated from any other labeled cell by at least 500 µm, were considered to be derived from the same progenitor cell, that is, to belong to the same clone. To appreciate their spatial distributions within the cortex, three dimensional reconstructions of the clusters were made using computer-aided microscope system for superposition of serial sections (Neurotrace, InterAction Co., Cambridge, MA). In view of the possibility of widespread dispersion of cortical clones as reported by Walsh and Cepko (34), we included in this analysis only hemispheres that contained no more than seven (typically two to three) widely spaced clusters of ␤-gal⫹ cells. Using this criterion, we believe that we have significantly reduced the risk of ‘‘lumping’’ error, that is, the possibility of including unrelated ␤-gal⫹ cells in a single cluster (see Ref. 20 for more extensive discussion). Following the identification of clusters of clonally re-

FIG. 3. Electron micrographs of labeled oligodendrocytes in layer V (A, B) and in the white matter (C) of the adult cortex. Although heavily stained, these cells exhibit features associated with oligodendrocytes: electron-dense nuclei with pronounced aggregates of heterochromatin. The cell shown in C is part of the clone encountered in the white matter and illustrated in Fig. 4G. A, ⫻8000; B, ⫻10,500; C, ⫻11,000. 422

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FIG. 4. Computer-aided reconstruction of glia clones identified in consecutive 100-µm-thick sections. (A–D) Examples of astrocyte clones in adult cerebral cortex of rats that received intraventricular injections of retrovirus at E14 (A), E16 (B), E17 (C), and E19 (D). (E–G) Reconstructions of oligodendrocyte clones from the cortices of animals injected with retrovirus at E17 (E), E19 (F), and E20 (G). Pia is at the top and white matter is at the bottom of each reconstruction. ⫻50.

FIG. 5. Computer-aided reconstructions of glia clones identified in consecutive 100-µm-thick sections through the cerebral cortex of rats that had received SVZ injections of retrovirus at different stages of postnatal life. An astrocyte clone (A) and a three-cell oligodendrocyte clone (B) that resulted from injections at P2. An astrocyte clone (C) and an oligodendrocyte clone close to the midline (D) in the corpus callosum (CC) that resulted from injections at P9. (E) A group of cells in the CC of an animal that had received a unilateral injection of retrovirus at P9 and sacrificed 3 days later. A few of the cells are on the side of the injection (left), while others are either on the midline or have crossed into the contralateral hemisphere. The presence of labeled cells in the CC of both hemispheres was common in the cortices of animals injected with retrovirus in the second postnatal week. Drawing of one section through part of the cortex of a rat injected at P14 showing the positions of two labeled cells, one in each hemisphere. Both cells were oligodendrocytes as suggested by the characteristic parallel processes shown in camera lucida drawings (F). ⫻50. 424

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FIG. 6. Photomicrograph of labeled cells in the CC of a rat injected with retrovirus at P9 and sacrificed 3 days later. These cells appear to be migrating from the side of the injection (left) to the contralateral hemisphere. Two of these cells (large arrow) are shown at higher magnification in the inset. ⫻300; inset, ⫻1200.

lated glial cells (see Results) for further analysis, camera lucida drawings were made of each constituent cell to reveal morphological details. In addition, a selected number of cells from a large number of clusters of glial cells were used for electron microscopical analysis. These cells were excised from the sections and mounted on Araldite stubs; ultrathin sections were cut through the cell and picked up on grids. The grids were then stained with 1% aqueous uranyl acetate and lead citrate and examined with the electron microscope. RESULTS

Morphological Features of ␤-Gal⫹ Glial Cells The light microscopic appearance of every ␤-gal⫹ cell was documented in camera lucida drawings. Neurons typically showed clear outlines of their somata and were considerably larger in size (8–20 µm in diameter) than cells identified as glia; some showed characteristic branching dendrites and axonal processes. Labeled glial cells typically had small (5–10 µm in diameter), round, or oval cell bodies, with only few, short stained processes. Some of these cells showed features characteristic of astrocytes (9, 15): processes with end feet on blood vessels or, less frequently, on the pial surface, and veil-like processes surrounding the soma (Fig. 1A). Others showed features of oligodendrocytes: darkly stained cell bodies, with few, short, thin processes often arranged in parallel (Fig. 1B). However, the lack of

process staining often exhibited by ␤-gal-labeled glial cells frequently impeded their phenotypic identity at the light microscopic level. In these cases, the ultrastructural appearance of ␤-gal⫹ glia enabled us to distinguish readily between astrocytes and oligodendrocytes (23, 24). Astrocytes can be distinguished from other cell types by the presence of glial filaments in a typically electron-lucent cytoplasm (Fig. 2). Another ultrastructural feature of this glial cell type is the irregular outline of their cell bodies and processes as they are closely molded to adjacent neuropil elements. Furthermore, many astrocytes are located adjacent to blood vessels and contribute processes to the subpial glia limitans. The vast majority of oligodendrocytes in the adult cerebral cortex may be classified as dark (23) (Fig. 3). The nucleus contains much heterochromatin, with large clumps aggregated beneath the nuclear envelope. The darkness of the cytoplasm is due in part to a high concentration of ribosomes and polysomal aggregates and in part to a granular ground substance between the organelles. Furthermore, the cisterns of the granular endoplasmic reticulum are more electron lucent than the surrounding cytoplasm, and the Golgi apparatus is generally prominent. Prenatal Injections The number and distribution of ␤-gal⫹ cells in the adult cerebral cortex following injections of retrovirus at each day of the last week of gestation (E14–E20)

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varied considerably among brains, probably as a result of (i) differences in the amount of virus injected in the lateral ventricles and (ii) the number of progenitor cells infected. These cells appeared either isolated in the cortex or as clusters of two or more cells. Cell clusters identified as clones were either neuronal or glial but, in very few cases, contained both neurons and glia. Brains injected at the earlier stages of corticogenesis (E14–E17) contained predominantly neuronal clones, whereas those injected with retrovirus toward the end of the gestation period (E18–E20) showed a significantly increased number of glia clones. Glia clones were encountered in the cortex of rats injected with retrovirus as early as the onset of corticogenesis (E14). At this and in the subsequent two stages (E15, E16), nearly all glia clones were composed of astrocytes; oligodendrocyte clones were rarely encountered. The brains of five of the rats injected with retrovirus at E14 contained 7 glia clones. All but one of these clones were composed of astrocytes spanning most of the cortical thickness. The number of cells per clone varied considerably, with the largest cluster comprising 94 astrocytes (Fig. 4A). The exception was a mixed clone comprising 23 astrocytes and 35 pyramidal neurons distributed in layers II–VI. A similar number of brains taken from rats injected with retrovirus at E15 were found to contain 9 glia clones. Eight of these clones contained only astrocytes, with the number of cells per clone ranging from 7 to 48, and the remaining clone comprised exclusively oligodendrocytes. Six of the astrocyte clones were observed in the gray matter, with cells scattered in most layers, and the other 2 appeared as tightly packed clusters in the subcortical white matter. The only oligodendrocyte clone also appeared as a discrete cluster of 46 cells in the white matter. There was also only 1 oligodendrocyte clone and 1 mixed clone, comprising astrocytes and pyramidal neurons, in the cerebral cortices of 14 brains that had received injections of retrovirus at E16. In these brains, 21 other clones consisted exclusively of astrocytes (Fig. 4B). These clusters, encountered in both the white and gray matter, contained between 7 and 55 cells. Starting with brains injected at E17, a somewhat increased number of oligodendrocyte clones appeared in the cortex. Twenty-nine glia clones derived from 12 brains were analyzed. Twenty-four of them contained only astrocytes and the other 5 comprised exclusively oligodendrocytes. The range of cells in the astrocyte clones was 4–96 cells per clone, and in the oligodendrocyte clones the range was 4–21 cells. Most astrocyte clones were encountered in the gray matter (Fig. 4C), although some had cells in both the gray and white matter. In contrast, only one of the oligodendrocyte clones was observed in the gray matter (layer IV; Fig. 4E); the remaining were located in the white matter. Similar proportions and dispositions of astrocyte and

oligodendrocyte clones were observed in the cortices of rats injected at E18. Toward the end of the gestation period, E19–E20, a significant increase in glia clones in the cortex was noted, while neuronal clones appeared less frequently. Twenty brains of animals injected with retrovirus at E19 contained numerous glia clones. Of the 65 such clones analyzed, 54 were composed exclusively of astrocytes (Fig. 4D) and 10 of oligodendrocytes (Fig. 4F); there was one mixed clone comprising 28 astrocytes and 2 pyramidal neurons. Again, most astrocyte clones were encountered in the gray matter while the majority of oligodendrocytes were found in the white matter. Astrocyte and oligodendrocyte clones appeared in similar proportions in the cortices of rats that had received injections of retrovirus at E20. Astrocyte clones (25 in number) ranged in size from 4 to 84 cells per clone, while the 8 oligodendrocyte clones encountered (Fig. 4G) were smaller, ranging from 3 to 13 cells per clone. To confirm an earlier suggestion (18) that the VZ contributes to the formation of the SVZ, two litters of rats injected with retrovirus at E19 were allowed to survive only until P7. Examination of sections through the cortices of these animals showed labeled cells not only in the cortex, but also in the SVZ, the area adjacent the dorsolateral part of the lateral ventricle in these young rats. These cells typically showed an elongated cell body and one thick, sometimes irregularly shaped process directed toward and terminating on the ventricular surface (Fig. 1C). Postnatal Injections Unilateral injections of BAG retrovirus were placed in the SVZ of rats 2, 7, 9, 11, and 14 days of age by means of a stereotaxic apparatus. These animals were allowed to survive until they were young adults (42 days), at which time sections through their cortices were examined for the presence of ␤-gal-labeled cells. Nearly all labeled cells appeared as discrete clusters distributed in the gray matter or in the subcortical white matter. The cellular composition and disposition of these clusters varied according to the age of the animal at the time of injection. Examination of the cerebral cortex of animals injected with retrovirus shortly after birth (postnatal day 2; P2) showed, in agreement with earlier studies (9, 14), discrete clusters of glial cells both in the gray matter and in the subcortical white matter. In the gray matter, astrocyte clones outnumbered oligodendrocyte clones (8 and 3, respectively), while the reverse was the case in the white matter (2 astrocyte and 4 oligodendrocyte clones). Astrocyte clones ranged in size from 4 to 12 cells per clone (Fig. 5A), whereas oligodendrocyte clusters contained between 3 and 8 cells (Fig. 5B). In the gray matter, astrocytes were often in close proximity to blood vessels with prominently stained end feet on

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these vessels. Oligodendrocytes showed little process staining in the gray matter, and for this reason electron microscopy was frequently used for their identification. However, in the white matter they showed the characteristic parallel processes in the orientation of the axons. Injections of retrovirus in the SVZ at the end of the first postnatal week (P7) showed a fairly even number of astrocyte and oligodendrocyte clones (12 and 11, respectively). However, 2 clones located in the middle layers of the cortex appeared to contain a mixture of astrocytes and oligodendrocytes. Astrocyte clones, found predominantly in the gray matter, contained 2–21 cells per clone, while the range for oligodendrocyte clones was between 2 and 23 cells. The majority of the clones at this stage were encountered in the white matter, with only 5 astrocyte and 2 oligodendrocyte clones present in the infragranular layers of the cortical gray matter. Twenty glia clones were analyzed in the cortices of brains injected at P9; 2 of them were mixed. Nearly all clones were encountered in the white matter of both the ipsilateral and the contralateral hemispheres and in the corpus callosum (Figs. 5C and 5D). Only 2 astrocyte clones and 1 oligodendrocyte cluster were seen in deep layer VI of the ipsilateral hemisphere. The observation of labeled cells in the contralateral hemisphere prompted us to investigate the time and the route by which glial cells make their way to that hemisphere. Toward this, we examined sections through the cortices of six rats, 3 days after retrovirus injections in the SVZ. In these sections we noted a number of labeled small cells as they appeared to migrate as a single file through the corpus callosum to the contralateral hemisphere (Figs. 5E and 6). Oligodendrocyte clones were the predominant glia clones (12 of the 14 clones analyzed) in the cortex of rats that received SVZ injections of retrovirus at P11. These clusters comprised 2–18 cells per clone and distributed entirely within the white matter of both hemispheres and in the corpus callosum. A similar distribution of oligodendrocyte clones was observed in brains injected at P14; no astrocyte clones were seen in these brains. It was interesting to note that these oligodendrocyte clones were distributed in the white matter medial to the level of the injection in the SVZ, but no further lateral than the SVZ of the contralateral hemisphere (Fig. 5F). DISCUSSION

The findings of this study, which traces the fate of glia progenitors present in the proliferative zones of the developing cortex of the rat, may be summarized as follows: (i) In the VZ there exist separate progenitor cells for astrocytes and oligodendrocytes as early as the onset of neurogenesis at E14. (ii) There is a very small

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population of oligodendrocyte progenitors in the VZ at the early stages of corticogenesis (2 of 39 glia clones in animals injected at E14–E16), increasing at later stages (28 of 110 glia clones following injections at E17–E20). (iii) Cells in the VZ give rise to progenitor cells that make up the SVZ in early postnatal life. (iv) These progenitor cells generate astrocyte and oligodendrocyte clones in the cortical gray and white matter shortly after birth, with astrocyte clones being in the majority. (v) At the end of the first postnatal week, astrocyte and oligodendrocyte clones are produced in roughly equal numbers, with the majority distributed in the subcortical white matter. (vi) In the second postnatal week, the proportion of oligodendrocyte clones increases continuously, all destined for the white matter of both the ipsilateral and the contralateral hemispheres including the corpus callosum. (vii) A small number of clusters (⬍10%) generated after SVZ injections contained both astrocytes and oligodendrocytes, suggesting that single SVZ cells can differentiate into both glial cell types. The finding of homogeneous glia clones in the adult cerebral cortex following retrovirus injections in the lateral ventricles of fetal rats is in agreement with earlier analyses of glial cell lineages in the forebrain (15, 16, 25). The present study further shows that glia progenitors undergo lineage restrictions earlier than previously reported, consistent with existing evidence for early gliogenesis in the forebrain (3). This analysis has also shown that oligodendrocyte progenitors are sparse in the VZ, particularly during the early stages of corticogenesis. A paucity of oligodendrocyte clones was also encountered in a previous study of cortical cell lineages in which only one of nine glia cell clones encountered in the mature cortex after retrovirus injections at E15–E16 was composed exclusively of oligodendrocytes (16). Oligodendrocyte clones increased in frequency in the later stages of gestation, but they still made up no more than 25% of all glia clones. These results clearly indicate that the VZ comprises predominantly neuronal progenitors that generate the pyramidal and nonpyramidal cell types of the cortex, astrocyte progenitors, and few cells dedicated to producing oligodendrocytes. However, they do not provide support for the reported presence of bipotential cells capable of generating neurons and oligodendrocytes (27, 35). What routes do glial cells use to move away from the proliferative zones in early cortical development to colonize the cortex and subcortical white matter? Analysis of astrocyte clones showed that these glial cells are organized in radially arranged subclusters, each comprised of several cells, often spanning most of the cortical thickness. This finding has prompted the suggestion that asymmetrical division and possibly both asymmetrical and symmetrical division are adopted by astrocyte progenitors to generate these cells, and that their distribution in radially arranged clusters sug-

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gests that they follow a pattern of radial migration using a scaffolding of radial glial processes similar to migrating cortical pyramidal neurons (19). If both these cell types migrate to the cortex along radial glia, then it is possible that groups of astrocytes and neurons, each derived from restricted progenitors, share the same scaffolding or closely positioned sets of radial glia processes that may guide them to closely spaced positions in the cortex. This may explain the presence of a small number of cell clusters composed of astrocytes and pyramidal neurons in the cortices of rats that had received prenatal injections of retrovirus (present results and Ref. 19). The presence in the adult brain of very few oligodendrocyte clones generated prenatally makes it difficult to draw definitive conclusions about their migratory routes to the cortex. Oligodendrocyte clones appear as discrete clusters in the gray matter and in the subcortical white matter. In the gray matter they are typically packed in the same layer or in two or three adjacent layers. Their distribution in clusters also suggests that they follow a radial migratory route to the cortex. However, analysis of glia clones in adult animals did not allow a distinction between clusters that migrated directly from the VZ and those derived from progenitors that migrated to the SVZ in the late embryonic stages. Thus, the presence of a greater number of glia clones in the brains of animals injected with retrovirus at the later stages of gestation does not necessarily imply the presence at these stages of more committed progenitors in the VZ. It is very likely that many of these clones arise from the SVZ in early postnatal life. Zerlin and colleagues (36) have examined the pattern of migration of cortical glial cells generated in the SVZ of the postnatal rat brain. They found that labeled glia, following injections of retrovirus in the SVZ, were often in radial arrangements and in close apposition to vimentin labeled radial glial processes. These findings strongly suggest that glia generated in the SVZ postnatally follow a radial migratory route to the cortical gray and white matter. Further indication is that the migration of these cells ceases by P14, which coincides with the collapse of the radial glial scaffolding (6, 21). However, there are indications that some glia may disperse in the cortex tangentially (see Ref. 36). Tangential pathways along axonal bundles are most likely used by astrocytes and oligodendrocytes of the white matter, especially those found in the contralateral hemisphere and in the corpus callosum. Injection of retrovirus in the postnatal SVZ resulted in the presence of a number of mixed glia clones (about 10% of the clusters analyzed). Although a number of investigations have not reported such mixed clones (14, 31), others have found that as many as 15% of the clones contained both astrocytes and oligodendrocytes (9). The presence in the adult cortex of a number of

discrete clusters containing both astrocytes and oligodendrocytes suggests that some SVZ cells retain a degree of developmental plasticity and are capable of producing both cell types. These observations, taken together with a large body of recent evidence, show that the SVZ of the postnatal brain may be seen as a mosaic of glia progenitors that give rise to cortical astrocytes and oligodendrocytes, of multipotential progenitors, of neuronal progenitors that produce a population of olfactory bulb neurons (12, 13), and of a pool of stem cells (22, 29). These cells have a capacity for self renewal and may represent a source for neurons and glia in the adult brain (4). ACKNOWLEDGMENTS I am grateful to Juliet Bower, Marion Cavanagh, Christina Danevic, Eva Franke, Brett Harris, Marina Mione, and Helen Perryman and especially to Peter Boardman for their contributions to various parts of the work. I also thank Stephen Davies for the help with the postnatal injections. The work was supported by the Medical Research Council.

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