In many parts of the central nervous system, the elongated processes of radial glial cells are believed to guide immature neurons from the ventricular zone to their sites of differentiation. To study the clonal relationships of radial glia to other neural cell types, we used a recombinant retrovirus to label precursor cells in the chick optic tectum with a heritable marker, the E. coli lacZ gene. The progeny of the infected cells were detected at later stages of development with a histochemical stain for the lacZ gene product. Radial glia were identified in a substantial fraction of clones, and these were studied further. Our main results are the following, (a) Clones containing radial glia frequently contained neurons and/or astrocytes, but usually not other radial glia. Thus, radial glia derive from a multipotential progenitor rather than from a committed radial glial precursor, (b) Production of radial glia continues until at least embryonic day (E) 8, after the peak of neuronal birth is over (∽E5) and after radial migration of immature neurons has begun (E6–7).

Radial glial and neuronal lineages do not appear to diverge during this interval, and radial glia are among the last cells that their progenitors produce, (c) As they migrate, many cells are closely apposed to the apical process of their sibling radial glia. Thus, radial glia may frequently guide the migration of their clonal relatives, (d) The population of labelled radial glia declines between E15 and E19–20 (just before hatching), concurrent with a sharp increase in the number of labelled astrocytes. This result suggests that some tectal radial glia transform into astrocytes, as occurs in mammalian cerebral cortex, although others persist after hatching. To reconcile the observations that many radial glia are present early, that radial glia are among the last offspring of a multipotential stem cell, and that most clones contain only a single radial glial cell, we suggest that the stem cell is, or becomes, a radial glial cell.

Radial glia are a prominent cell type in many parts of the vertebrate central nervous system (Ramon y Cajal, 1911; Levitt and Rakic, 1980; Edwards et al., 1990). Their cell bodies are confined to the ventricular zone but their processes span the width of the neural tube: a short basal process runs from the cell body to the ventricular surface, and a long apical process ascends to the pia where it terminates in an endfoot. In cerebral cortex, where they have been studied most intensively, radial glia appear very early in development and then disappear perinatally (Ramón y Cajal, 1911; Schmechel and Rakic, 1979a; Levitt and Rakic, 1980; Misson et al., 1988b; Edwards et al., 1990). The shape of the radial glia and their transient nature are consistent with the idea that one of their functions is to guide immature neurons from their birthplace in the ventricular zone to their ultimate destination in the cortical plate. Ultrastructural studies in vivo (Rakic, 1972; Gadisseux et al., 1990), and studies of neuronal-glial interactions in vitro (reviewed in Hatten, 1990), have provided support for this hypothesis.

Although considerable information is available about the structure and fate of radial glia (reviewed in Edwards et al., 1990; Misson et al., 1991; Cameron and Rakic, 1991), relatively little is known about their origins and early development. Among questions that remain unanswered are: What is the nature of the progenitor that gives rise to radial glia? Is it committed to the production of radial glia or is it multipotential? Is there any clonal relationship between radial glia and the neurons that migrate along them? In addition, the timing of radial glial production is unclear -histological studies in cortex indicate that some appear early, but it is not known how late into the period of neurogenesis radial glia continue to be produced (Schmechel and Rakic, 1979b; Misson et al., 1988a). More is known about late stages of embryogenesis, during which radial glial processes defasciculate, become increasingly branched, undergo systematic distortions in trajectory (Misson et al., 1988a; Gadisseux et al., 1989; Edwards et al., 1990; Takahashi et al., 1990) and eventually disappear; several lines of evidence suggest that this disappearance reflects a transformation of radial glia into astrocytes (Ramón y Cajal, 1911; Choi and Lapham, 1978; Schmechel and Rakic, 1979a; Pixley and deVellis, 1984; Voigt, 1989; Cullican et al., 1990).

Here, we have used retrovirus-mediated gene transfer (Sanes et al., 1986; Price et al., 1987) to analyze the lineage, differentiation, and fate of radial glia in the chicken optic tectum. The optic tectum is a useful system for such studies because of its regular structural organization and because of its accessibility at early embryonic stages. In previous work, we used recombinant retroviruses as clonal markers to study the genealogical relationships and migratory paths of tectal neurons and astrocytes (Gray et al., 1988; Galileo et al., 1990; Gray and Sanes, 1991). We showed that several types of neurons and two types of astrocytes derive from a common precursor in the tectum, and that many neurons migrate along bundles of radial glial processes to their laminar destinations. To extend this analysis, we have now used similar methods to demonstrate clonal relationships among neurons, astrocytes and the radial glia that guide their migration.

Injection of virus

Fertile White Leghorn chicken eggs were obtained from SPAFAS (Roanoke, IL), and incubated on their sides at 37.5°C in a Humidaire hatcher (New Madison, OH). For lineage analysis, a retroviral concentrate was pressure-injected into the right tectal ventricle of staged (Hamburger and Hamilton, 1951) embryos. The virus used, LZ10, is a recombinant Rous sarcoma virus in which the structural genes from wild-type virus are replaced with the E. coli f-galactosidase (ZacZ) gene. Virus was produced, collected, concentrated, and injected as previously described (Gray et al., 1988; Galileo et al., 1990). Following injection, eggs were sealed with transparent tape and returned to the incubator until they reached an appropriate developmental stage.

Fixation

Embryos were killed at E10, 12, 15, or 19–20 (approximate stages 36, 38, 41, and 45, respectively; hatching is at E20-21). Whole brains were removed from E10–15 embryos, and fixed by immersion in 2% paraformaldehyde plus 0.03% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4. E19–20 embryos were anesthetized with ether and perfused intracardially with the same fixative before dissection. Tecta to be stained for lacZ were fixed for 1–2 h at room temperature. Those used for immunohistochemistry or Dil labeling were fixed for up to 1 week at 4°C.

X-gal histochemistry

Fixed tecta were sectioned perpendicular to their long axes at 100–200 μm on a Vibratome (Pelco, Redding, CA). Sections were placed directly into a solution of 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal), potassium ferrocyanide, and potassium ferricyanide, prepared as described previously (Gray et al., 1988). Sections were incubated overnight at room temperature, washed with phosphate-buffered saline (PBS; 150 mM sodium chloride, 15 mM sodium phosphate, pH 7.3), dried onto subbed slides and mounted with polyvinyl alcohol (Mowiol, Hoechst Celanese Corp., Sommerville, NJ).

Immunohistochemistry

Fixed tecta were sunk overnight in 30% sucrose in PBS, then frozen in 2-methylbutane that had been cooled by liquid nitrogen. Sections were cut on a cryostat at 15–20 μm, mounted on subbed slides, air-dried, and frozen at —20°C until they were stained. Sections were incubated with primary antibodies overnight at 4°C, washed with PBS and stained 1 h at room temperature with secondary antibodies. Slides were mounted in 90% glycerol, 10% PBS, supplemented with paraphenylenediamine. Some sections were counterstained with bisbenzamide (1 μg/ml; Hoechst 33258; Sigma, St. Louis, MO) to reveal nuclei. Primary antibodies used were R5, a mouse monoclonal IgM that selectively stains radial glia (Drager et al., 1984; generously provided by Ursula Drager, Harvard University); H5, a mouse monclonal IgG that reacts with the same antigen as R5 (Herman et al., 1991); and a rabbit anti-lacZ antiserum, generated in our laboratory using purified E. coli /-galactosidase (Sigma, St. Louis, MO) as the immunogen. Secondary antibodies were rhodamine-conjugated goat-anti-mouse IgG+IgM and fluorescein-conjugated goat-anti-rabbit IgG (Organon Teknika-Cappel, Malvern, PA, and Boehringer-Mannheim, Indianapolis, IN).

Dil

Dil (Molecular Probes, Eugene, OR) was dissolved at 2.5 mg/ml in absolute ethanol as suggested by Honig and Hume (1986), and injected into the ventricles of fixed E10 tecta using a 1 ml tuberculin syringe and a 25 gauge needle. Tecta were incubated at room temperature for ∽1 week, then sectioned on a Vibratome at 50–100 μm and examined with rhodamine fluorescence optics. The fine crystals that formed in the ventricle often stained only isolated radial glial fibers.

Determination of number of radial glia

A ∽2 mm × 2 mm piece was cut from the center of six E15 and seven E19 tecta that had been fixed in 2% paraformaldehyde. The pieces were stained with R5 antibody as described above, then mounted whole on slides. For each tectum, 5 fields of glial endfeet (or terminal glial shafts) were photographed, and the number of endfeet per unit area was determined from micrographs. To normalize for surface area, three tecta each from E15 and E19 embryos were sectioned completely at 200 μm on a Vibratome (Peleo, Inc.), and the surface area of the tecta was calculated from the lengths of the pial surface in each section.

Identification of radial glia

To study cell lineage in the optic tectum, we inject a recombinant Rous sarcoma virus that bears the lacZ gene into the ventricle of the embryonic mesencephalon. The viral genome is incorporated into the DNA of dividing infected precursor cells, and the lacZ gene is transcribed and translated. LacZ is readily detected in the progeny of infected precursors using either a histochemical stain or antibodies to lacZ.

When we injected virus into the tecta of embryonic day (E) 3–5 chicken embryos and examined them on E15, we found that some of the labelled cells were elongate in shape: their cell bodies lay in the ventricular zone, and a single long, varicose process extended from each cell body to the pia (Fig. 1A,B). Short lateral processes were occasionally present as well,-but the apical process was unbranched. These cells were similar in size and shape to radial glia revealed by either of two methods (Vanselow et al., 1989; Gray and Sanes, 1991): uptake of dil from crystals applied to the ventricular surface (Fig. 1 C-E), and immunohistochemical staining with the monoclonal antibodies R5 (Fig. 1F) or H5 (Fig. 2B), both of which recognize an intracellular antigen in radial glia (Herman et al., 1991). Importantly, the periodic thickenings of the apical processes observed in X-gal stained cells (Fig. 1A and B) were also evident in dil-stained cells (Fig. 1C and D), indicating that these swellings were cytoplasmic varicosities rather than separate adherent cells. This point (which was previously made by Ramon y Cajal, 1891) was confirmed by staining with bis-benzamide to reveal DNA, and showing that the varicosities were anucleate (data not shown).

Fig. 1.

Morphology of radial glia in the optic tectum. Radial glia were labelled by X-gal following infection at E5 (A) or E7 (B), by Dil applied to the ventricle (C, D, E), or by staining with R5 (F). Tecta are from E10 (C,D) or E15 (A,B,E,F) animals. Bars, 50 μm (A-D); 100 μm (E,F).

Fig. 1.

Morphology of radial glia in the optic tectum. Radial glia were labelled by X-gal following infection at E5 (A) or E7 (B), by Dil applied to the ventricle (C, D, E), or by staining with R5 (F). Tecta are from E10 (C,D) or E15 (A,B,E,F) animals. Bars, 50 μm (A-D); 100 μm (E,F).

Fig. 2.

Sections double labelled with anti-lacZ and H5, a monoclonal antibody that labels radial glia. (A,C) LacZ-positive fibers, (B,D) same fields labelled with H5. The lacZ-positive fibers are stained by H5, demonstrating that they are the apical processes of radial glia. The tectum was injected with retrovirus at E4 and stained at E15. Bars, 25 μM.

Fig. 2.

Sections double labelled with anti-lacZ and H5, a monoclonal antibody that labels radial glia. (A,C) LacZ-positive fibers, (B,D) same fields labelled with H5. The lacZ-positive fibers are stained by H5, demonstrating that they are the apical processes of radial glia. The tectum was injected with retrovirus at E4 and stained at E15. Bars, 25 μM.

To confirm the identification of the elongated lacZ-positive cells as radial glia, we prepared cryostat sections from virus-infected tecta, and double-labelled them with antibodies to lacZ plus R5 or H5. Fig. 2 shows that the lacZ-positive apical processes were also H5-positive. Together, these results demonstrate that some of the cells infected by retrovirus on E3-5 were progenitors of radial glia.

Clonal relatives of radial glia

For lineage analysis, tecta were injected with LZ10 at E3-4, fixed at E15, and stained histochemically for lacZ. Clusters of lacZ-positive cells were identified as clones by criteria that have been detailed elsewhere (Gray et al., 1988, 1990). Approximately one third of the clones analyzed (for example, 35/106 from one set of tecta injected on E3, and 72/218 from a set injected on E4) contained cells that were unambiguously classifiable as radial glia on morphological grounds: they had a soma in the ventricular zone and an apical process that extended at least one third of the distance to the pial surface. These clones were studied further.

Fig. 3 illustrates several features of radial glia-containing clones. First, some clonal relatives of radial glia were clearly neurons, as determined by their round nuclei, abundant cytoplasm, and the characteristic arrangements of their processes (Fig. 3D and E). Many other cells were likely to be neurons as well, but their small size and poorly stained processes precluded their positive identification. Nonetheless, it is likely that most progenitors that give rise to radial glia give rise to neurons as well. Second, many clones contained astrocytes. These could be identified on the basis of their small, irregularly shaped cell bodies, and highly branched filamentous processes (Fig. 3C). (Immunohistochemical confirmation of the identity of lacZ-positive cells as neurons and astrocytes has been reported previously; see Galileo et al., 1990.) Third, many clones contained both neurons and astrocytes as well as radial glia (Fig. 3A,B). Together, these results demonstrate that individual tectal progenitors labelled on E3-4 can give rise to neurons, astrocytes, and radial glia.

Fig. 3.

Clonal relatives of radial glia. (A,B) Camera lucida drawings of clones marked by infection on E3 (A) or E4 (B), and examined at E15. Cells with neuronal and astrocyte morphologies are found in association with the single radial glial cell in each clone. Arrows in A and B indicate cells showm in C-F. (C) A cluster of astrocytes. (D) Fusiform neurons. (E) A large pyriform neuron. (F) A radial glial cell body and its proximal process. Bars, 50 μm (A,B); 20 μm (C-F). P, pia; TP, tectal plate; 13, lamina 13 (also called stratum griseum centrale); IZ, intermediate zone; VZ, ventricular zone; V, ventricular surface.

Fig. 3.

Clonal relatives of radial glia. (A,B) Camera lucida drawings of clones marked by infection on E3 (A) or E4 (B), and examined at E15. Cells with neuronal and astrocyte morphologies are found in association with the single radial glial cell in each clone. Arrows in A and B indicate cells showm in C-F. (C) A cluster of astrocytes. (D) Fusiform neurons. (E) A large pyriform neuron. (F) A radial glial cell body and its proximal process. Bars, 50 μm (A,B); 20 μm (C-F). P, pia; TP, tectal plate; 13, lamina 13 (also called stratum griseum centrale); IZ, intermediate zone; VZ, ventricular zone; V, ventricular surface.

Finally, most radial glia did not have other radial glia as clonal relatives (Fig. 3A and B). For example, in one set of 56 radial glia-containing clones from tecta infected at E3, there were 44 (79%) with only a single radial glial cell, 9 (16%) with two, and 3 (5%) with four. In animals infected at E4, 96% (54/56) of radial glia-containing clones bore one radial glia, and the remainder had two each. We showed previously that clones marked on E3 are composed of one or a few radial strands of cells, whereas nearly all clones marked at E4 or later consist of only a single strand (Gray et al., 1988). Each strand contains ∽10–15 cells by E8, when cell division is nearly complete, and may represent the progeny of a single stem cell (Gray and Sanes, 1991). Of the 14 clones that contained >1 radial glial cell following injection at E3 or E4, only 2 contained >1 radial glial cell within a single strand. Thus, our results are consistent with the possibility that most stem cells produce no more than one radial glial cell.

Time of radial glia production

Having found that radial glia and neurons were both present in clones marked by injections on E3–4, we asked how long radial glia continue to be produced and whether the radial glial and neuronal lineages eventually diverge. Animals were injected with virus at E3, 4, 5, 6, 7, 8, 10, and 12, then sacrificed and analyzed at E15. Numerous radial glia were labeled after injection at E3-8, but few lacZ-positive cells of any type were seen in tecta injected on E10-12. Although this result may indicate that radial glia are not born after E8, it is also possible that virus was unable to infect progenitors efficiently at late stages, for example, because the developing basal lamina may impede access of virus to cells in the ventricular zone. Thus, we do not know when production of radial glia ceases, but we can conclude that it proceeds at least until E8, which is after the peak of neuronal generation is over (∽E5; LaVail and Cowan, 1971) and after radial migration of neurons has begun (∽ E6– 7; Gray and Sanes, 1991).

Fig. 4 shows typical radial glia-containing clones generated from E5 and E8 infections. As expected, the average number of cells per clone was smaller at E8 than at E5 and smaller at E5 than at E3 (compare with Fig. 3). As in tecta injected earlier, nearly all clones that contained radial glia included only a single radial glial cell (30/30=100%, 60/61=98%, and 25/26=96% following injection at E5, E7 and E8, respectively). Furthermore, even in small clones, which consisted of only 3– 4 cells, some of the clonal relatives of radial glia were identifiable as neurons (Fig. 4A; see also Figs 4B and 7A). Thus, we obtained no evidence that the neuronal and radial glial lineages diverge.

Fig. 4.

Radial glial clones from tecta injected at E5 (A) or E8 (B) and stained for lacZ at E15: camera lucida drawings. (A) A clone consisting of a single radial glial cell with two associated cells, one of which is a large multipolar neuron. In many clones, multipolar neurons migrate tangentially from the radial array (Gray and Sanes, 1991), but this one has apparently not done so. (B) A clone that consists of one radial glial cell plus one associated cell (arrow) that is probably a small neuron. Fewer cells are associated with the radial glia in clones marked at these stages, compared with clones marked at E3 and E4 (Fig. 3), but neuronal and glial lineages have not diverged. Bars, 50 μm. Abbreviations, as Fig. 3.

Fig. 4.

Radial glial clones from tecta injected at E5 (A) or E8 (B) and stained for lacZ at E15: camera lucida drawings. (A) A clone consisting of a single radial glial cell with two associated cells, one of which is a large multipolar neuron. In many clones, multipolar neurons migrate tangentially from the radial array (Gray and Sanes, 1991), but this one has apparently not done so. (B) A clone that consists of one radial glial cell plus one associated cell (arrow) that is probably a small neuron. Fewer cells are associated with the radial glia in clones marked at these stages, compared with clones marked at E3 and E4 (Fig. 3), but neuronal and glial lineages have not diverged. Bars, 50 μm. Abbreviations, as Fig. 3.

The presence of labeled radial glia in tecta injected at E7 and E8, late in the period of neuronal birth (LaVail and Cowan, 1971), raised questions about the relative timing of radial glial and neuronal production. If production of radial glia peaked before that of neurons, radial glia should make up a smaller percentage of the total population of lacZ-positive cells with successively later ages of infection. If, however, most radial glial were produced after most neurons were born, the percentage of labeled radial glia in late-infected tecta should increase and the percentage of neurons should decrease. To distinguish these alternatives, we classified lacZ-positive cells in a set of tectal sections from each age of injection as “radial glia”, “astrocytes”, or “neurons and others”. Neurons were not counted separately because, as noted above, many cells that were likely to be neurons could not be distinguished unambiguously; in fact, most of the cells in the third category are likely to be immature neurons. As shown in Fig. 5, successively later injections label populations of cells that are increasingly enriched in radial glia. This trend is evident whether or not astrocytes are included in the totals -i.e., whether the frequency of labeled radial glia is calculated with respect to all labeled cells, or to only the population of presumptive neurons. Thus, radial glial production continues even after neuronal production has begun to wane. Taken together with the lineage analysis described above, these results indicate that radial glia are likely to be among the last cells produced by their multipotential progenitors.

Fig. 5.

Percentage of lacZ-positive cells that are radial glia following infection at various ages. All labeled cells were counted in 34– 42 sections from each of 5– 10 animals per stage, and the percentage of radial glia was calculated with respect to presumptive neurons (open circles) or to all labeled cells (closed circles). By either measure, radial glia are preferentially labelled by later infections.

Fig. 5.

Percentage of lacZ-positive cells that are radial glia following infection at various ages. All labeled cells were counted in 34– 42 sections from each of 5– 10 animals per stage, and the percentage of radial glia was calculated with respect to presumptive neurons (open circles) or to all labeled cells (closed circles). By either measure, radial glia are preferentially labelled by later infections.

Radial glia as guides for clonal relatives

In E8– 12 tecta, when cells are actively migrating from the ventricular zone to the tectal plate, most of the radially migrating cells are closely associated with fascicles of radial glial processes (Gray and Sanes, 1991), suggesting that radial glia act as migratory guides in tectum, as they are thought to do in mammalian cortex (Rakic, 1972; Gadisseux et al., 1990). In tecta examined at E15, when migration is largely complete, clonal relatives of a radial glial cell were often found in close association with that cell’s process, sometimes apparently contacting it (e.g., Fig. 3). Taken together, these two observations raised the possibility that tectal neuroblasts might use sibling radial glia as migratory guides.

To test this idea, we analyzed a set of clones at E10, during the peak of radial migration. For this experiment, tecta were injected on E4 (rather than on E3, as in Gray and Sanes, 1991), to generate relatively small clones (single radial strands; see above). In these tecta, we found numerous clones that contained radial glia. The majority of these clones consisted of tight radial arrays of cells in which a single radial glial cell was accompanied by radially migrating cells whose somata and processes were closely apposed to the radial glial process (Fig. 6A). In other cases, individual clones contained several cells with somata in the ventricular zone and intertwined processes that extended apically (Fig. 6B); it is likely that at least one cell in each of these clones was a radial glial cell and that the full extent of its apical process was obscured by the opposed processes of its migrating siblings. In clones of both types (Fig. 6A and B) several cells were frequently arranged along the length of the same process, both in the intermediate zone and in the tectal plate. The more apical cells in clones were sometimes displaced tangentially from the radial glial process (Figs 6A and 7A) consistent with the possibility that they had reached their laminar destinations and ceased migration. In addition, in some clones, cells within an individual radial strand appeared to be aligned along two or three adjacent fascicles, only one of which contained a labeled radial glia; there was not, therefore, a complete restriction of migrants to a single fascicle (see also Gray and Sanes, 1991). Nonetheless, in the majority of clones that contained both radial glial cells and radially migrating cells, the majority of the migrants were aligned along the fascicle that contained the process of the labeled radial glia. These results raise the possibility that radial glial cells guide their clonal relatives from the ventricular zone to their destinations in the tectal plate.

Fig. 6.

Guidance of radially migrating cells by sibling radial glia. Tecta were injected at E4 and stained at E10, during the peak of radial migration. (A) Typical clone, in which one radial glial cell is clearly identifiable. The migrating cells (arrows) are tightly associated with the glial process, one in the intermediate zone and two in the tectal plate. A fourth sibling, the most superficial, is displaced laterally by ∽ 20 μm. (B) A larger clone with several cells in the ventricular zone that have ascending processes; at least one of these is likely to be a radial glial cell. Bars, 50 μm. Abbreviations, as Fig. 3.

Fig. 6.

Guidance of radially migrating cells by sibling radial glia. Tecta were injected at E4 and stained at E10, during the peak of radial migration. (A) Typical clone, in which one radial glial cell is clearly identifiable. The migrating cells (arrows) are tightly associated with the glial process, one in the intermediate zone and two in the tectal plate. A fourth sibling, the most superficial, is displaced laterally by ∽ 20 μm. (B) A larger clone with several cells in the ventricular zone that have ascending processes; at least one of these is likely to be a radial glial cell. Bars, 50 μm. Abbreviations, as Fig. 3.

Fate of radial glia

To follow the fate of radial glia over time, we injected groups of tecta at the same stage, and then fixed and stained subgroups at various times between E10 and E20. Between E10 and E15, as noted above, radial glia were abundant, and they were accompanied by differentiating cells that could sometimes be identified as immature neurons (Figs 3, 4, 6 and 7A). At E19-20, on the other hand, a few clones were encountered that contained radial glia (Fig. 7B), but the vast majority of clones contained no cells whose somata lay in the ventricular zone. To quantify the apparent loss of radial glia, we calculated the percentage of all lacZ-positive cells that were radial glia as a function of stage of sacrifice for three experiments in which tecta were injected at E3, E7, or E8, respectively. As shown in Table 1, there is a sharp decline in the population of labeled radial glia during the period between E10 and E19, and this decline does not depend greatly on the age of infection.

Table 1.

Percentage of radial glia at different stages of sacrifice

Percentage of radial glia at different stages of sacrifice
Percentage of radial glia at different stages of sacrifice
Fig. 7.

LacZ-positive clones from E10 and E19 tecta. (A) A single radial glial cell associated with an immature neuron (arrow), marked by injection on E7 and examined at E10. The neuron appears to be parting from the radial glial process. (B) A single radial glial cell forms the core of a large clone that was marked by injection on E3 and examined at E19. Clones of this sort are rare, as few lacZ-positive radial glia can be found at this age. Bars, 50 μ m. Abbreviations, as Fig. 3.

Fig. 7.

LacZ-positive clones from E10 and E19 tecta. (A) A single radial glial cell associated with an immature neuron (arrow), marked by injection on E7 and examined at E10. The neuron appears to be parting from the radial glial process. (B) A single radial glial cell forms the core of a large clone that was marked by injection on E3 and examined at E19. Clones of this sort are rare, as few lacZ-positive radial glia can be found at this age. Bars, 50 μ m. Abbreviations, as Fig. 3.

There are several possible reasons for the decline in lacZ-positive radial glia. One possibility is that lacZ may not be expressed well in radial glia at late embryonic stages. Another is that radial glia may die during this period. While we cannot exclude either of these alternatives, three observations provide indirect evidence for a third possibility: that at least some radial glia transform into astrocytes. First, between E15 and E19-20, clusters of astrocytes appeared throughout the tectal layers; occasionally these clusters were arranged radial to one another. The appearance of astrocytes correlated with the decline in radial glia. Second, in tecta examined before E19, cells were often present that were intermediate in form between radial glia and astrocytes. These cell types were especially abundant at E15, which is approximately when the first immunohistochemically identifiable astrocytes appear in the tectum (Linser and Perkins, 1987). Fig. 8 illustrates the different radial-glia-form cell types seen in the tectum at E12, 15, and 19, and suggests a possible progression in the transformation of radial glia into astrocytes. Third, if radial glia transform into astrocytes, one would expect the ratio of radial glia to astrocytes to be constant at any single time of sacrifice, regardless of the age of injection. In fact, there was no systematic variation of this ratio with age of injection. For example, in animals analyzed at E15, the ratio of radial glia to all glia (radial glia plus astrocytes) was 0.59,0.45, 0.55, and 0.38 after infection at E4, E5, E7, and E8, respectively. Finally, it is important to note that these findings are in agreement with evidence that radial glia in mammalian cortex transform into astrocytes (see Discussion).

Fig. 8.

Intermediate cell types suggest that radial glia transform into astrocytes. Camera lucida composites of glia from tecta analyzed at E12 (A), E15 (B) and E19 (C). (A) Radial glia are numerous and astrocytes rare at E12. (B) Several cell types seen at E15 that may represent the progressive transformation of radial glia into astrocytes. They are arranged here in a hypothetical sequence of transitional forms, suggested by studies in mammalian cerebral cortex (e.g., Schmechel and Rakic, 1979; Misson et al., 1988b). (C) Clusters of astrocytes that appear as the numbers of labelled radial glia decline. Bar, 100 μm. Abbreviations, as Fig. 3.

Fig. 8.

Intermediate cell types suggest that radial glia transform into astrocytes. Camera lucida composites of glia from tecta analyzed at E12 (A), E15 (B) and E19 (C). (A) Radial glia are numerous and astrocytes rare at E12. (B) Several cell types seen at E15 that may represent the progressive transformation of radial glia into astrocytes. They are arranged here in a hypothetical sequence of transitional forms, suggested by studies in mammalian cerebral cortex (e.g., Schmechel and Rakic, 1979; Misson et al., 1988b). (C) Clusters of astrocytes that appear as the numbers of labelled radial glia decline. Bar, 100 μm. Abbreviations, as Fig. 3.

If the decline in lacZ-positive radial glia is representative of the general behavior of all radial glia, one should see a decline in the total population of radial glia between E15 and 19. However, Vanselow et al. (1989) saw abundant radial glia at the time of hatching. We therefore compared the numbers of radial glia present at E15 and 19, by labeling pieces of fixed tecta with R5, and counting the glial endfeet that lie just beneath the pia, as described in Materials and methods. After normalizing for changes in surface area we found a decline in radial glial endfeet of 14% between E15 and 19. This difference is less than would be predicted from the data in Table 1, although the discrepancy could be explained if (a) subpial radial glial branching increased with age, as has been seen in rodents (Edwards et al.,1990) (b) the transformed radial glia were replaced by production of new radial glia from unmarked progenitors, (c) transitional forms (Fig. 8) maintained pial endfeet after their somata left the ventricular zone, or (d) a decline in lacZ expression at late ages exaggerated the numbers of radial glia that were lost. Despite the discrepancy, the fact that there is a decrease in radial glial numbers between E15 and 19 is consistent with the idea that some radial glia are transforming into astrocytes.

We have used retrovirus-mediated gene transfer to label radial glial cells and their clonal relatives in the chicken optic tectum. Our results show that radial glia arise from a progenitor that also generates neurons and astrocytes, but generally does not produce more than one radial glial cell. Radial glia are produced until at least E8, which is after many neurons are born and after migration has commenced. During migration many neurons associate with clonally related radial glia. Subsequent to migration, at least some radial glia may turn into astrocytes, although many others remain in the tectum at least until hatching.

Lineage

In previous studies of cell lineage in the optic tectum, we demonstrated that several types of neurons and two kinds of astrocytes can arise from a common precursor (Gray et al., 1988; Galileo et al., 1990; Gray and Sanes,1991) In those studies, in which clones were analyzed at E19 (just before hatching), we identified few lacZ-positive radial glia. We therefore suspected that most radial glia were generated early in development, before we had injected virus to mark progenitors. Subsequently, we were struck by the abundance of lacZ-positive radial glia in a set of clones analyzed at E15. Eventually, we learned that many lacZ-positive radial glia disappear between E15 and E19, although a few remain. By combining studies at E15 and E19, we were able to compare radial glia-containing clones with those we already knew to contain neurons and astrocytes. Together with our previous work, the data presented here indicate that radial glia derive from the same types of progenitors that give rise to several other tectal cell types. We do not know whether these progenitors are all of a single type, or whether several distinct types of progenitors exist that generate overlapping sets of cells. However, our results suggest that neuronal and radial glial lineages do not diverge, even at late stages of neurogenesis.

An additional feature of radial glial lineage is their likely transformation into astrocytes after their role as migratory guides has ended. Such a transformation was orginally postulated to occur in chicken spinal cord by Ramón y Cajal (1911), on the basis of transitional cellular forms observed in Golgi stained material. Ramón y Cajal (1911), Choi and Lapham (1978), and Schmechel and Rakic (1979a) later extended this idea to the mammalian cortex. More recently, antibodies that stain mammalian radial glia, the transitional forms, and astrocytes, have been used to demonstrate a relationship among these cells in vivo (Levitt and Rakic, 1980; Pixley and deVellis, 1984; Misson et al., 1988a; Voigt, 1989; Takahashi et al., 1990) and in vitro (Cullican et al., 1990). Finally, Voigt (1989) labelled radial glial cells with an intracellular dye at an early stage, and later identified astrocytes containing the label, thus providing the most direct evidence of transformation to date. Together, these studies provide substantial support for the idea of radial glial transformation.

Three lines of evidence presented here indicate that a similar transformation occurs in tectum. First, the number of lacZ-positive radial glia declines during the period that lacZ-positive astrocytes appear. Second, cells whose morphology is intermediate between radial glia and astrocytes appear in the tectum as the numbers of radial glia decline. Finally, the ratio of radial glia to astrocytes remains fairly constant, regardless of age of infection. On the other hand, a substantial number of radial glia remain in the tectum of hatchlings, indicating that many do not transform into astrocytes, or do so only posthatching.

The results of our clonal analysis permit us to evaluate several alternative models of radial glial lineage (Fig. 9). Perhaps surprisingly, our results appear to exclude, for the tectum, the scheme that is currently favored for mammalian cerebral cortex -i.e., that neuronal and radial glial lineages diverge early, with radial glia giving rise to many (if not all) astrocytes, and a separate class of neuroepithelial cells giving rise to neurons (Fig. 9A). The idea that radial glial and neuronal lineages diverge early in cortex is supported by observations on primates by Levitt et al. (1980,1981, 1983). They showed that two populations of mitotically active cells can be distinguished in the cortical ventricular zone on the basis of immunoreactivity for the astrocyte-specific intermediate filament, glial fibrillary acidic protein (GFAP), and that the GFAP-positive subpopulation corresponds in large part to radial glia. In light of evidence that radial glia transform into (GFAP-positive) astrocytes (see above), they argued that the GFAP-negative progenitors give rise to neurons (reviewed in Cameron and Rakic, 1991). Misson et al. (1988a, 1988b, 1991) have made a parallel set of observations in murine cortex, using the monoclonal antibodies RC1 and RC2 to distinguish radial glia from other neuroepithelial cells, and to follow the transformation of radial glial into astrocytes. Our retrovirus-based clonal analysis in murine cortex (Luskin et al., 1988), and that of Price and Thurlow (1988) in rat, have provided direct evidence that neurons and astrocytes have distinct progenitors, and thus indirectly support the model of Rakic and others. However, clonal analyses that include radial glia themselves have not yet been reported.

Fig. 9.

Models of radial glial lineage. (A) Scheme currently favored for mammalian cortex, in which radial glia (RG) give rise to astrocytes (A), and neurons (N) arise from separate progenitors (NB) (see Cameron and Rakic, 1991). (B) Scheme A modified to fit data that tectal progenitors give rise to both neurons and astrocytes (see Galileo et al., 1990). (C) Scheme in which radial glia are early-born progeny of a multipotential stem cell. (D) Scheme suggested by data in this paper, in which a radial glia cell is one of the last progeny of a multipotential stem cell. See text for details.

Fig. 9.

Models of radial glial lineage. (A) Scheme currently favored for mammalian cortex, in which radial glia (RG) give rise to astrocytes (A), and neurons (N) arise from separate progenitors (NB) (see Cameron and Rakic, 1991). (B) Scheme A modified to fit data that tectal progenitors give rise to both neurons and astrocytes (see Galileo et al., 1990). (C) Scheme in which radial glia are early-born progeny of a multipotential stem cell. (D) Scheme suggested by data in this paper, in which a radial glia cell is one of the last progeny of a multipotential stem cell. See text for details.

In tectum, radial glia, neurons, and astrocytes all frequently descend from a single precursor, and radial glia may be among the last cells to be produced. Thus we can exclude not only cortex-like schemes (Fig. 9A) but also modified schemes in which the radial glial lineage diverges early from a neuron-glia lineage (Fig. 9B), or in which radial glia are among the first-born progeny of a multipotential progenitor (Fig. 9C). Instead, our results favor schemes such as that shown in Fig. 9D, in which: 1) a single multipotential progenitor produces neurons, astrocytes, and radial glia; 2) radial glia are among the last-born progeny of their progenitor; 3) progenitors marked at E3 or later produce only a single radial glia; and 4) some but not all radial glia transform into astrocytes or astrocyte precursors late in embryogenesis.

The progenitor in Fig. 9D is drawn as a stem cell -a cell which undergoes asymmetric divisions to produce another mitotically active stem cell plus a postmitotic daughter. This feature is based on indirect evidence that proliferative and stem cell divisions occur sequentially in tectum. First, progenitors divide symmetrically (proliferatively) to produce equivalent, horizontally displaced daughters that populate the ventricular zone. Subsequently, these cells use a stem cell mode of division to produce radial arrays of postmitotic neurons and glioblasts (Gray et al., 1988,1990). If this scheme is correct, our new results raise the possibility that the stem cell is itself an immature radial glial cell. This idea would provide a way to reconcile the notion that many radial glia are present early with the observations that many tectal radial glia are generated late, and would account for the result that <5% of the clones marked on E4 or later contain >1 radial glia. In fact, mitotic divisions of differentiated radial glia have been observed in adult avian telencephalon (Alvarez-Buylla et al., 1990), denervated adult amphibian optic tectum (Stevenson and Yoon, 1981), and embryonic mammalian cerebral cortex (Schmechel and Rakic, 1979b; Levitt et al., 1983; Misson et al., 1988b). In cortex, it has been supposed that mitoses of radial glia generate additional radial glia and, ultimately, astrocytes. In adult birds, radial glia have been suggested to be progenitors of neurons, based on the coincidence of sites of mitotic activity with sites at which neurons are known to arise (Alvarez-Buylla et al., 1990). Finally, Fredricksen and McKay (1988) used immunohistochemical similarities between neuroepithelial cells and radial glia to suggest that radial glia are stem cells. Thus, it is possible that radial glia are mitotically active stem cells in many parts of the nervous system, but give rise to different types of progeny in different regions.

One implication of the notion that radial glia are stem cells (or that stem cells become radial glia once they have ceased to produce other cell types) is that each clone should contain a radial glial cell. However, only ∽35% of the clones marked by injection at E3 or E4 and analyzed at E15 contain unambiguously identifiable radial glia. Careful examination of the remaining clones shows, however, that another ∽40% contained cells that were likely to be radial glia, but did not meet the strict criteria we used in classifying cells for clonal analysis -for example, some of these contained a soma in the ventricular zone with an apical process that extended less than one third of the way to the pia, and others contained “transitional forms” (see Fig. 8) presumably derived from radial glia. Interestingly, inclusion of these “likely” radial glia did not greatly increase the number of clones that contained >1 radial glial cell. Therefore, few clones contained >1 radial glia-like cell per strand, only about 25% contained no such cells, and some of the latter might be accounted for by incomplete expression or detection of lacZ. In short, it is possible that the majority of tectal stem cells are, or become, radial glial cells.

Radial glia as migratory guides

Current interest in radial glia centers in large part on their role in guiding the migration of neurons to their laminar destinations. Two of our results may be informative in this regard. The first is that many radial glia are generated late in the period of neurogenesis. Neuronal birth starts around E3 and peaks at E5-6 (LaVail and Cowan, 1971). Newly born cells stack up in the ventricular zone until about E6, when a minority of cells begins a tangential migration along axons in the marginal zone. Few radially migrating cells penetrate the marginal zone until ∽E7, when the first radially migrating cells can be found in association with radial glia (Gray and Sanes, 1991). It is possible that the late generation of radial glia (or their differentiation from stem cells; see above) opens a radial migratory path, thus terminating the recruitment of new neuroblasts to the tangential pathway and initiating radial migration to form the tectal plate.

The second relevant observation is that there is a regular association between sets of clonally related migrants and their sibling radial glial cell. Most migrating tectal cells migrate in association with bundles of radial glial fibers (Gray and Sanes, 1991). Clonally related cells generally migrate along one or a few bundles of radial fibers, resulting in radially oriented clones (Gray et al., 1988; Gray and Sanes, 1991). Here, we have shown that the bundle that guides a clone of cells frequently includes the apical process of a clonally related radial glial cell. In that a number of radial processes may be available to migrating cells within the bundle, we cannot conclude that neuroblasts migrate only along clonally related glial cells. Lacking ultrastructural studies on this point, it is premature to speculate on whether the association reflects only proximity or some form of chemical recognition. Regardless of the mechanism, however, the association of clonally related radial glia and radially migrating cells may be partially responsible for generating the arrays of cells that became functionally interconnected (Jassik-Gerschenfeld and Hardy, 1984; Rakic, 1988), as development proceeds.

We thank Jeanette Cunningham and Robin Morris-Valero for assistance. This work was supported by grants from the McKnight Foundation and the National Institutes of Health.

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