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Development 101. 409-419 (1987) Printed in Great Britain © The Company of Biologists Limited 1987 Review Article 409 Retrovi ruses and the study of cell lineage JACK PRICE Laboratory of Embryogenesis. National Institute of Medical Research. Mill Hill, The Ridgeway, London NW7 1AA, UK Key words: retrovirus. cell lineage, virus. RNA. haematopoiesis. mouse embryo, histochemical marker, nervous system Introduction Retroviruses and retroviral vectors In this review, I want to discuss a new way of tackling an old problem. The problem is how to mark a cell such that its developmental capacity can be assayed. The solution I want to consider is gene transfer using retroviruses. There are many ways of marking cells, but a genetic marker has a number of obvious advantages. It is indelible, heritable and need not damage a cell or distort its development. However, it is not always easy to introduce a genetic marker into a cell, especially if the number of marked cells as well as the precise time in development at which the marker is introduced need to be controlled. Retroviruses have a number of properties that make them versatile and powerful tools in the study of development. Their principal advantages stem from the fact that they are a naturally evolved system for transferring genes into cells of a host animal (Fig. 1). As a consequence, this transfer is highly efficient (unlike most artificial means) and also highly accurate in that a faithful copy of the retroviral genome is integrated into the host cell chromosome. Furthermore, the retrovirus itself carries the nucleic acid sequences required to direct the host cell in the transcription of the retroviral genes. This means that in many situations the virus can be relied upon to express itself without recourse to further genetic manipulation. On the other hand, viruses can be engineered to provide other promoter elements should they be required (Wagner, Vanek & Vennstrom, 1985; Stewart, Vanek & Wagner, 1985; Emerman & Temin, 1984, 1986). The structure of the retroviral genome has evolved with the retroviral genes located in the middle of the genome, flanked by sequences called the long terminal repeats (LTRs). The significance of the LTRs is that they contain all the sequences required in cis for the integration and expression of the retrovirus. This organization is principally a reflection of the mechanism of replication that retroviruses have evolved (Varmus, 1982), but it is fortunate from the point of view of the molecular biologist because it means that, in principle, any gene could be introduced in place of the retroviral genes, and the virus could still infect a cell, integrate and transcribe that cloned gene. (The genetic engineering of retroviruses is essentially the same as cloning in plasmids and mostly uses the techniques of conventional molecular biology.) Such expression of cloned DNA in retroviruses has now been shown to work in a number of instances, as will be described below. Of equal importance has been the development of methods for the packaging of recombinant viruses For several years now, molecular biologists have been using a variety of gene transfer techniques (see Gordon & Ruddle, 1985, for review), the most familiar of which to embryologists is probably the microinjection of DNA into the mouse pronucleus as a means of generating transgenic mice (Gordon et al. 1980; Wagner, Stewart & Mintz, 1981; Harbers, Jahner & Jaenisch, 1981; Brinster et al. 1985). However, it is not obvious how the methods of molecular biology, which are primarily in vitro techniques, can be applied to problems of gene transfer in vivo. Retroviruses may be a way out of this predicament. The work I shall discuss in this review represents the initial studies from a small number of laboratories, in which retroviruses have been applied to the study of cell lineage. In concentrating on this aspect of retroviral technology, I am ignoring several other interesting applications of retroviruses (such as insertional mutagenesis and gene therapy) and also excluding much of the molecular biology of the structure and function of retroviruses. (Good reviews of this area exist in any case: see Varmus, 1982; Coffin, 1985; Bernstein, Berger, Huszar & Dick, 1985.) 410 / . Price into infectious particles (Fig. 2). This is done by transfecting a plasmid that contains the engineered virus into what is termed a packaging cell line (Mann, Mulligan & Baltimore, 1983; Watanabe & Temin, 1983; Cone & Mulligan, 1984; Sorge, Wright, Erd- Reverse transcription ^ ^ _ DNA DNA man & Cutting, 1984). This permanent cell line makes the retroviral gene products but makes no viral genomic RNA that is capable of forming a retroviral particle. In other words, it has all the ingredients necessary for making a virus with the exception of packageable retroviral RNA. The engineered virus, however, once introduced into the cell will form such an RNA, which consequently is packaged and released by the transfected packaging cells. In this manner, the tissue culture supernatant from these cells constitutes a permanent supply of high titre virus. Viruses of this type, that encode foreign genes but not the endogenous retroviral genes, are usually termed retroviral vectors. They are infective in the Packaging cell line, produces retroviral proteins (required for packaging) but no packageable RNA .0. Engineered retroviral genome TRANSFECTION in plasmid neo SELECTION Clones isolated and expanded Fig. 1. The retroviral life cycle. This figure is a schematic diagram of the life cycle of a typical wild-type retrovirus. The retroviral particle is adsorbed onto the cell plasma membrane by the binding of its envelope surface glycoproteins to a specific surface receptor. Following fusion, the retroviral genomic RNA passes into the cytoplasm, and is reverse transcribed into DNA, gains entry into the nucleus and as a proviral circle integrates randomly into the host cell chromosomal DNA. The integrated provirus acts as typical chromosomal DNA in that it is inherited by both daughter cells whenever the cell divides. The provirus is also transcribed and the retroviral genes are translated, using the cell's normal machinery. The assembly of a new retroviral particle completes the life cycle. The genomic retroviral RNA transcript comes together with the retroviral gene products and buds off to form a new free retroviral particle. The above description relates to a wild-type retrovirus. A retroviral vector of the type considered in this review is deficient in one important aspect of this life cycle. The retroviral vector is infective in the same manner as a wild-type virus, it is reverse transcribed and integrates as normal, and (depending on the construct) is transcribed and translated. However, because it does not have the retroviral genes, it cannot form the retroviral gene products required to assemble new retroviral particles. This is of crucial importance for lineage studies because it means that an infected cell cannot pass virus on to neighbouring, uninfected cells. Only its daughter cells will inherit the provirus from an infected cell. \ Packaged, engineered retroviral vector released by cells into culture medium Fig. 2. The production of a retroviral vector using a packaging cell line. When a retroviral vector is constructed that does not code for the endogenous retroviral genes, some way must be found to package the engineered retroviral genome into a retroviral particle. The most popular method of doing this is to use a packaging cell line. This is a cell line that has been transfected with a retroviral genome that encodes all the retroviral genes and so makes all the retroviral gene products. However, this retroviral genome is deficient in a sequence that is required for packaging. Consequently, the genomic RNA transcribed from this provirus cannot be packaged even though the retroviral gene products are present. One can think of a packaging cell as a cell waiting to package appropriate RNA into viral particles but with no suitable RNA of its own to package. To package a retroviral vector, the cells are transfected with a plasmid that contains an engineered retrovirus. Typically a selectable marker such as the neo gene is included in the construct, so that clones of transfected cells can be selected and isolated. Such clones package the engineered virus and release this packaged retrovirus into the culture medium. Therefore, the cell lines derived from these clones provide a permanent supply of the engineered retrovirus. Retroviruses and the study of cell lineage same manner as wild-type viruses, but, on infecting a cell, they cannot complete the wild-type life cycle shown in Fig. 1, because they do not contain the endogenous retroviral genes required to package an RNA. A variety of studies has now been done using retroviral vectors of this type to infect cells in culture. In the earliest experiments, the cloned genes used were predominantly those that encode selectable marker genes such as the Herpes simplex thymidine kinase (tk) gene or the Tn5 neo gene, which infers G418 resistance on eukaryotic cells (see Bernstein et al. 1985 for references). But more ambitious constructs have also begun to appear and the expression of preproparathyroid hormone (HeUerman et al. 1984), granulocyte-macrophage-colony-stimulating factor (Lang et al. 1985), the polymeric immunoglobulin receptor (Deitcher, Neutra & Mostov, 1986) and fibronectin polypeptides (Schwarzbauer, Mulligan & Hynes, 1987) have all now been reported. Retroviruses as lineage markers Once a retrovirus has infected a cell, it integrates into the host cell genome, so that the pro virus is inherited by all the progeny of that cell. Hence, the clone of cells derived from the infected cell is genetically marked. Moreover, when the provirus integrates, it does so randomly, so that each integration site is unique. Consequently, on infection each cell (and subsequent clone) is given a genetic label. So if the host cell DNA is cut with a restriction enzyme, and the DNA fragments separated electrophoretically and hybridized with a probe recognizing the viral sequences, the fragment of DNA from each clone that contains the provirus will be unique and of a characteristic size, which will distinguish it from any other such fragment. This manner of marking clones with a retrovirus provides one way in which they can be used to study cell lineage. One of the advantages of this approach is that expression of the viral genome is not required, the presence of the integrated provirus being sufficient to recognize the clone. However, a potential disadvantage of the approach is that in situations where there is retroviral expression, infected cells can generate new retroviral particles and so spread the virus in a horizontal fashion, thereby obscuring any clonal analysis. The most elegant way of avoiding this problem is to use the retroviral vectors described above, which are replication defective and, therefore, cannot spread horizontally to other cells. I will return to this approach to lineage later in this review. However, another solution to the problem is to study systems in which virus does not express. For example, the 41 ] Muloney murine leukaemia virus (MoMLV) does not express in cells of the preimplantation mouse embryo (Jaenisch et al. 1975). It appears that the proviral DNA becomes methylated (Stewart, Stuhlman, Jahner & Jaenisch, 1982), although it is not clear that this is the primary reason for the lack of expression (Gautsch & Wilson, 1983; Niwa, Yokata, Ishida & Sugahara, 1983). Consequently, studies of lineage in the early mouse embryo are possible using wild-type MoMLV retrovirus, and these experiments are described below. This manner of recognizing clones - studying band sizes on Southern blots — has been productive in two principal areas of research into cell lineage. These are haematopoiesis and the early development of the mouse embryo. Haematopoiesis The development of blood cells was in many ways an obvious place to begin applying gene transfer techniques to the study of cell lineage. It is one of a few vertebrate systems where a considerable amount was already known about cell lineage relationships, largely as a result of experiments using nonretroviral chromosomal markers (see Quesenberry & Levitt, 1979, and Till & McCulloch, 1980 for reviews). Also, techniques already existed for the removal and culture of mouse bone-marrow cells and their subsequent introduction into an irradiated syngeneic host. Probably the main drive to research in this area is the hope that the haematopoietic system might be amenable to gene therapy. The lineage of the haematopoietic system is possibly better understood than that of any other mammalian system. It has been clear for some time that there is a stem cell (CFU-S) that has the ability to generate the entire myeloid lineage (Till & McCulloch, 1961). This stem cell is self replicating (Simnovitch, McCulloch & Till, 1963) and is believed to generate committed progenitor cells, which can give rise to particular differentiated cell types (Lewis & Trobaugh, 1964; Curry & Trentin, 1967). Several workers have shown that bone marrow cells can be infected in vitro with retroviral vectors such that a proportion of the stem cell population becomes marked. On reintroduction into an irradiated host, these cells can successfully repopulate both myeloid and lymphoid cell compartments (Joyner, Keller, Phillips & Bernstein, 1983; Miller et al. 1984; Williams et al. 1984; Hock & Miller, 1986). Although in the earliest studies only a small proportion of the stem cells was labelled, techniques have more recently been refined so that now it is possible to label close to 100% of the stem cells (Eglitis, Kantoff, Gilboa & Anderson, 1985; Dick et 412 /. Price al. 1985; Lemischka, Raulet & Mulligan, 1986). This is done generally by coincubating the bone marrow cells with high titre viral producer lines and by pushing the predominantly quiescent stem cells into mitosis with growth factors, or by treating the donor animal with 5-fluorouracil prior to removing the marrow. This drug kills dividing cells and so pushes stem cells into division to compensate for the lost populations. Consequently, there is a higher proportion of stem cells in the total bone marrow population. The increased division of the stem cells is significant because only dividing cells seem able to integrate virus. The increased proportion of stem cells is significant because normally these cells are a tiny minority of the total population and as such are quite difficult to study. A question of some interest was whether the retroviral technique could be used to label the pluripotential stem cell that gives rise to both myeloid and lymphoid derivatives. It is now clear from a number of studies that such cells can be identified with this technique (Williams et al. 1984; Dick et al. 1985; Keller, Paige, Gilboa & Wagner, 1985; Lemischka, Raulet & Mulligan, 1986). The importance of this observation lies not so much in proving the existence of a pluripotential haematopoietic stem cell (there has been good evidence in this regard for many years (Wu, Till, Siminovitch & McColloch, 1968; Abramson. Miller & Phillips, 1979)) but in demonstrating the relative ease with which it is possible to label such cells and follow their fate during development. With these better marking techniques, more ambitious experiments have become possible and the initial results are promising. For example, Lemischka and his colleagues were able to analyse how the contribution of marked stem cells to various cell compartments varied over periods of months in the same animal (Lemischka et al. 1986). Interestingly, they found that each cell compartment was made up of the derivatives of relatively few clones, but that over a period of 2-3 months the contribution shifted as certain clones apparently disappeared from the differentiated cell populations whereas others, previously undetected, now appeared. This is not, of course, a normal animal, recovering as it is from radiation and bone marrow transplant. Nonetheless, these results suggest that a limited number of stem cells contributes to the haematopoietic system at any one moment. The system is, in other words, oligoclonal and, with time, stem cells cease to contribute to the pool of differentiated cells and are replaced. It was also possible in the same study to analyse the contribution that marked clones make to various organs and anatomical locations in repopulated mice (Lemischka et al. 1986). Consequently, the authors observed stem cells that had a broad potential in terms of the cell types to which they could give rise, yet seemed to be restricted in the tissues they repopulated. A clone could contain, for instance, splenic but not thymic T cells, or bone marrow but not peritoneal macrophages. (The authors do not, however, describe exactly which combinations of cell types and locations could be clonally derived and which could not.) These data should, of course, be interpreted with care; just because a stem cell only populates certain compartments does not prove that its fate was determined and that it could not have populated other compartments. Nonetheless, this observation raises the possibility that haematopoietic cells can become restricted in terms of the anatomical compartments that they populate as well as in the types of blood cell to which they give rise. If this conclusion proves to be correct, it will be interesting to see how it influences conventional models of haematopoietic cell lineage. These studies of haematopoiesis are still at an early stage and they are, I think, clearly going to influence more than just ideas on lineage. Another obvious direction is to introduce genes into haematopoietic stem cells with a view to analysing the effect this has on their development. Oncogenes, growth factors and various surface receptors all come to mind in this regard, as do the various clinically important genes that, it is hoped, may become candidates for gene therapy. (See, for example, Ledley, Grenett, McGinnis-Shelnutt & Woo, 1986; Williams, Orkin & Mulligan, 1986; Jolly et al. 1986.) Lineage analysis in the early mouse embryo A similar strategy to that described for haematopoiesis has also been applied to the study of lineage in the preimplantation mouse embryo. Embryos can be infected at early stages then introduced into pseudopregnant foster mothers for implantation. This approach has been used by a number of workers (Jaenisch, Fan & Croker, 1975; Rubenstein, Nicolas & Jacob, 1986; Soriano & Jaenisch, 1986; Stewart, Schuetze, Vanek & Wagner, 1987). Essentially, this is similar to other methods of generating chimaeras but it is notably less invasive, requiring no injection of DNA or aggregation of embryos. Alternatively, embryonic carcinoma (EC) cells (Stewart et al. 1982) or embryo-derived stem cells (ES or EK cells) can be infected in culture (Evans, Bradley, Kuehn & Robertson, 1985; Robertson, Bradley, Kuehn & Evans, 1986) and then used to form chimaeras using established methods. In this manner, the retroviral sequences are introduced into a subpopulation of the embryonic cells. These techniques have been used for a number of purposes including insertional mutagenesis, which Retroviruses and the study of cell lineage will not be dealt with here (but see Schneike, Harbers & Jaenisch, 1983; Jaenisch etal. 1985; King etal. 1985; Robertson, 1986; Robertson et al. 1986). For the study of lineage, though, this approach has been taken up by Soriano & Jaenisch (1986) as a means of analysing early events in the mouse. They infected embryos at the 4- to 16-cell stages by cocultivating them for 24 h with MoMLV producer cells. After introducing the embryos into a foster mother, they allowed normal development to proceed and then were able to ask in which lineages the progeny of marked blastomeres had appeared. For example, they asked whether clones were shared between embryonic and extraembryonic tissues, or between somatic and germ line; and within the somatic tissues, whether clones were represented in all tissues or restricted to a subset of organs? They reached some interesting conclusions. They found very little evidence for a lineage common to both placenta and embryo at this stage: of 52 proviral integration sites examined at 14 days post coitum (E14), 25 were found in the embryo, 27 in the placenta and only 3 in both. In mosaic animals that were allowed to progress to adulthood, the authors were able to assess to what extent a marked blastomere that had contributed to one organ, the liver for example, had also contributed to others. They found that in the vast majority of cases, a blastomere that had contributed to any one organ of the eight or nine that they analysed, had also contributed to the rest. They were able to quantify this observation. By densitometric scanning of a Southern blot of DNA from each tissue, the authors could estimate the intensity of any given proviral band relative to DNA from tissues of animals that were heterozygous for the provirus (that is, all cells of the tissue carried one copy of the provirus). In other words, if the control cells were considered to have a value of 1, i.e. one copy per cell in the entire tissue, then a value of, say, 0-5 would mean that half of the cells of that tissue had a copy of that particular provirus (and hence were derived from the blastomere that had been infected). This relative value (which the authors want to call 'molarity') gives an estimate of the proportion of the cells in any tissue that are derived from an infected blastomere. They observed that for any given proviral integration this value was the same in all tissues. That is, the derivatives of marked blastomeres made up a roughly equal proportion of all organs. This was true for almost all the proviruses analysed (2 exceptions out of 35). These were, in other words, fine-grained mosaics. This must mean, as the authors conclude, that considerable stem cell mixing and division must occur prior to the setting apart of tissue anlagen. By breeding chimaeric mice and analysing the appearance of proviruses in the offspring, the authors 413 were able to show not only that labelled blastomeres contributed to the germ line, but also that some of these had failed to contribute to somatic tissues. This suggests that at the time of proviral integration, some blastomeres were set aside in the germ cell (as opposed to somatic cell) lineage. Many of these data fit with those obtained from chimaeras generated by the aggregation of preimplantation embryos (Gardner, 1978; Rossant, 1984). For example, it has been observed previously that blastomeres at such stages can contribute to the somatic lineage but not to the germ cell line. The observation that blastomeres before the 64-cell stage can contribute to the germ line but not the somatic lineage is slightly more surprising as it implies that the germ line is set aside at an earlier stage than has previously been thought. Certainly, other techniques have identified precursors common to both these lineages as late as 5 or 6 days post coitum (McMahon, Fosten & Monk, 1983; Gardner et al. 1985). As noted above, Soriano and Jaenisch have quantified their Southern blot data by comparing the contributions made by different clones to a number of mosaic animals. When all these values were compared, the authors found that the lowest value that appeared was 0-12, even though they estimated that they could have detected as low as 0-06. Since a value of 0-12 is roughly one eighth as a fraction, this means that if a clone contributes to a tissue, it contributes at least an eighth of the cells of that tissue. The authors interpret this finding to mean that eight cells are set aside to make the entire embryo. There are, I think, two immediate problems with this interpretation. First, one would like some evidence that this value of 0-12 has some real significance. Simply the fact that this is the lowest value found in this particular experiment cannot be taken to mean that it is significant biologically without some evidence that it is the invariant minimum. Basically, this is a statistical problem and should be treated as such. Second, as has already been noted by Rossant (1986), one difficulty with this interpretation, even taken at face value, is that it is not clear when these eight cells would be set aside. A problem with the technique (as Soriano and Jaenisch point out) is that although infection takes place quite quickly, it seems by comparison with tissue culture studies that the provirus can remain in the cell for some time before it actually integrates. Stewart et al. (1982) have estimated that with MoMLV infections of F9 cells, this lag can be up to 2 days. This means that although the infection took place at the 4- to 16-cell stage in Soriano and Jaenisch's experiments, integration (and hence 'marking') may have taken place as late as the 64-cell stage. So even if the 8-cell theory is correct, 414 J. Price one wonders which eight cells are being discussed and when are they put aside. Retroviral vectors encoding histochemical markers Apart from the intrinsic value of their findings, the studies of haematopoiesis and embryonic lineage described above have indicated the value of retroviruses as lineage markers. Furthermore, I think it is fair to say that they have been successful while having employed retroviruses to only a fraction of their potential. All the lineage studies discussed so far have had a noticeable limitation. Because the method of detection takes the form of a Southern blot, the technique is limited to analysing relatively large populations of cells. This limitation has two immediate consequences. First, small clones cannot be detected. Second, it is not known which cells within a population belong to the marked clone, so that, if the population contains more than one cell type, it is not possible to say whether the marked cells included only one or many cell types. Unfortunately, these limitations exclude a number of the more interesting types of lineage study that might be undertaken. For example, in the nervous system, it would be interesting to know whether neurones and glial cells are derived from the same set of progenitor cells, or whether separate glial and neuronal precursors are set aside early in development. The inability to answer questions of this type is a considerable handicap in developmental biology, not just in the study of neural development. It is particularly frustrating in trying to move from a descriptive to a mechanistic analysis of developmental events. The reason for this is clear from the example given above. Unless the timing of different decisions and the order in which crucial decisions are made are known, it is difficult to decide which of the potential interactions and influences are likely to be significant. Indeed, much of the renewed interest in lineage in vertebrates stems from the envy with which those studying vertebrates look upon the successes in the invertebrate field. The concept of 'compartments' in insects is well known now and needs no repeating here. But it is noteworthy that the understanding of lineage relationships in invertebrates has also been crucial in unravelling developmental events where lineage per se plays a minor role in determining fate. One example of this is the development of the insect eye where it has been shown that lineage plays no part in determining cell type (Ready, Hanson & Benzer, 1976; Lawrence & Green, 1979). This finding has helped focus attention on the later cell-cell interactions that are apparently the significant events in determining how cells form an ommatidium (Tomlinson, 1985; Tomlinson & Ready, 1986; Lebovitz & Ready, 1986). Fortunately, there are other ways in which a retrovirus can be employed to study lineage. It is possible to use a vector that not only integrates and genetically marks a clone, but also expresses an inserted gene. If the product of this gene can be identified histochemically, then even a single cell expressing this product can be recognized. This gives the advantage of being able to study small clones and the ability to identify precisely which cells within a structure are part of a clone. There are so far two published reports of this approach being successfully applied. In one of them, Sanes and his collaborators at the Pasteur Institute have used a retroviral expression system to look at postimplantation lineages in the mouse embryo (Sanes, Rubenstein & Nicolas, 1986). The other was work in which I have been involved in collaboration with David Turner and Connie Cepko of Harvard Medical School. We have investigated lineage in the nervous system of the rat (Price, Turner & Cepko, 1987; Turner & Cepko, 1987). The two approaches are similar. In both cases, retroviral vectors based on MoMLV were constructed that expressed the bacterial lacZ gene, which encodes for the enzyme /3-galactosidase. However, the two viruses were constructed differently. In our BAG virus, the lacZ gene comes under the control of the endogenous retroviral promoter and enhancer elements, whereas Sanes et al. used an internal SV40 early promoter to drive the gene. /S-galactosidase has a number of obvious advantages as a marker. It has a number of convenient assays which make the quantification of expression straightforward. Similarly, there are a variety of monoclonal and polyclonal antibodies available against the /3-galactosidase protein. Most valuably, there is a quick, sensitive histochemical method to detect cells that are expressing the enzyme. This uses the substrate X-gal and can be used on cultured cells or tissue sections or even tissue whole mounts, to stain cells expressing /3-galactosidase. In addition to these features, it seems likely that embryonic cells could express high levels of /3-galactosidase without it interfering with normal development. Of course, that is difficult to prove, for such interference might be in ways that go unnoticed. However, in other systems, high levels of/3-galactosidase expression have occurred with no apparent perturbation (Lis, Simon & Sutton, 1983; Hiromi, Okamoto, Gehring & Hotta, 1986; Goring etal. 1987) and, in the studies considered here, there was no evidence of abnormality (see references below). Retroviruses and the study of cell lineage Postimplantation lineages In the study by Sanes et al. (1986), virus was injected into the mouse embryo in utero between 7 and 11 days post coitum (E7 to Ell). At this stage of development, it is not possible to see the embryo proper through the uterine wall because of the decidual tissue. The only hope is to inject the virus into the amniotic cavity or yolk sac and so expose the embryo to virus. There is also a limit to the amount of virus that can be injected into such a small structure and it is impressive that these workers could get sufficient virus into the embryo to infect any cells at all. Despite this limitation, they were able to study the lineage of a number of different tissues. In particular, they describe a series of clones in the visceral yolk sac and in the skin. When they injected virus at E7, the clones they found upon subsequent examination of the yolk sac contained cells from the mesothelial layer, capillary endoderm and fibroblasts. In other words, all the cell types in the visceral yolk sac that are mesodermally derived were found together in clones. Cells of the visceral endoderm were not labelled. When virus was injected at E9, the majority of clones contained only capillary endothelial cells and fibroblasts. Only one clone contained mesothelial cells and it had no other cell type. They also found one clone containing fibroblasts alone. The authors interpret these data to suggest that at E7 there is a common progenitor which gives rise to the three mesodermal derivatives, capillary endothelium, fibroblasts and mesothelium. By E9, however, it seems that progenitors exist that give rise to either fibroblasts and capillary endothelial cells, or to mesothelium. This suggests that a common progenitor that generates all three cell types gives rise by E9 to two progenitors with a more restricted potential. In the skin, the story is similar. From their E9 injections, the authors found clones of epidermal cells and, in four out of five cases, these clones also contained cells of the periderm. (This is the outermost layer of cells found covering embryonic but not adult skin.) Interestingly, these clones could contain cells of both the ordinary epithelium and of the hair germ, the structure that develops into a hair. When the clones from E l l injections were analysed, three out of four clones contained epidermis but not periderm and the fourth contained periderm but not epidermis. The conclusion was, therefore, that at E9 a bipotential progenitor exists which gives rise to cells that by E l l can make only epidermis or periderm but not both. In the yolk sac and skin, therefore, Sanes and his colleagues were able to define a progressive restriction in the cell types to which ancestral cells 415 could give rise. They were also able to delineate to some extent when these restriction events took place. These studies are important for a number of reasons. First, they have obviously gone some distance towards sorting out some of the lineage relationships of the tissues they have examined. But also, this study proves the feasibility of retroviral lineage studies at this stage of development. What remains to be seen is whether the lineages of less accessible tissues can be studied. One might expect it to be more difficult at, say, E9 to get virus into some of the mesodermal or endodermal embryonic structures as this would require infection of cells within embryo itself. It remains to be seen whether or not this is possible. Lineage studies in the nervous system The retroviral approach that has been taken to lineage analysis of the nervous system is similar to that of Sanes etal. (1986) except that we concentrated on the later stages of development in an effort to resolve questions regarding the final generation of cell diversity (Price et al. 1987; Turner & Cepko, 1987). Interestingly, whereas the study of Sanes et al. has indicated lineages that diverge early in development, the results in the nervous system have shown, somewhat surprisingly, that some types of progenitor cells exist that can give rise to an array of cell types even very late in development. This was shown most clearly in the rat retina. Some cell types of the rat retina are generated embryonically, but most rod photoreceptors and some amacrine cells, bipolar cells and Miiller glia are generated postnatally. If the BAG virus is injected into the retinas of new-born rats and their retinas analysed histochemically in adulthood for clones that express /J-galactosidase, then one finds clones containing all these cell types (Price etal. 1987; Turner & Cepko, 1987). It can also be shown that the number of clones observed is proportional to the amount of virus injected and that, if the viral titre is calculated in this fashion, it differs from the in vitro titre on 3T3 cells by a factor of only four (Turner & Cepko, 1987). The clones were small (two or three cells on average) as would be expected so late in development, and by far the majority of clones contained only rods - again as might be expected given that this is the main cell type being generated at this time. The interesting question is what are the lineage relationships of these retinal cell types? Turner and Cepko have analysed hundreds of clones and not only did they find mixed clones containing more than one cell type, but they also observed clones containing every possible combination of rods, amacrine cells, bipolar cells and Miiller glia, except that no clones 416 J. Price contained all four cell types. However, given the size of clones and the low frequency of occurrence of some of the cell types such 'four cell type' clones would be expected to be rare. The simplest interpretation of these data is that each progenitor in the neonatal rat retina can give rise to all cell types. A less-likely interpretation is that there are numerous types of progenitor cell, each of which can give rise to a different combination of the four cell types. In either case, it seems that a retinal progenitor cell can give rise to two quite different cell types up to its final division. This is very reminiscent of the situation in the insect retina referred to earlier, but runs somewhat contrary to what is thought to occur in the mammalian central nervous system where separate glial and neuronal lineages are thought to be generated quite early (see Levitt, Cooper & Rakic, 1981; Rakic, 1983). This latter view has been supported in recent years by the work of Raff and his colleagues which has shown the existence in the rat optic nerve of a bipotential progenitor which can give rise to one type of astrocyte and oligodendrocyte but not to other types of astrocyte or any neuronal cells (Raff, Miller & Noble, 1983). This implies the existence in the optic nerve, at least, of progenitors that have the potential to make less than the full complement of neural cell types. None of these data are contradictory, of course; the developmental strategy that the animal applies in the retina might be quite different from that used in the brain. It is now necessary to apply the /?-galactosidase retroviral marker technique to other regions of the CNS; and that is currently being tried. Sanes et al. (1986) reported finding some clones in the brain although no clonal analysis was reported. We have infected embryonic cortical cells in culture and, judging primarily by morphological criteria, found clones of glial cells containing both protoplasmic and fibrous types of astrocytes (Price et al. 1987, and unpublished observations). It is, however, too early yet to interpret these results with assurance. In all of the above discussion, I have ignored one possible problem with the /3-galactosidase vector studies. In the early mouse embryo and haematopoietic studies, clonality was recognized by the fact that a proviral integration marked a unique site in the genome. In the histochemical studies using the /S-galactosidase vectors, this was not possible. Discrete areas of stained cells were interpreted as being clones. There is, however, the possibility that the 'clone' was the result of two neighbouring cells being infected with virus. In practice, the possibility of two clones being superimposed is slight as, in most cases, the use of low viral titres ensures that an infection is a rare event. For example, Turner and Cepko typically used titres of virus that gave around one hundred clones (average two cells each) over an entire retina. Similarly, in our experiments with cortical cells in vitro (Price et al. 1987) a culture with about 105 cells would have perhaps five or ten marked clones. Nonetheless, an individual result should be viewed cautiously and, in all the experiments cited here, most types of clones were found several times. The only exceptions were some of the clones found by Sanes et al. (1986) in their embryonic injections where, presumably, clones were rare. In the cited studies on the retina, each clone could be positively identified as such by its strict radial arrangement (an observation of interest in itself) so that even clones quite close together could generally be resolved. So clonality is not a problem unless one believes that there is some predisposition for infective events to cluster and there is no evidence for that. It is likely that the studies described in this review are just the foretaste of good things to come. The potential of retroviruses is only beginning to be explored; there are many more adventurous retroviral constructs than those described here. Consequently, it should now be possible to establish the lineage relationships of many cells in the vertebrate using some combination of the techniques reviewed here. This is an exciting possibility. 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