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

In this review, I want to discuss a new way of tacklingan old problem. The problem is how to mark a cellsuch that its developmental capacity can be assayed.The solution I want to consider is gene transfer usingretroviruses. There are many ways of marking cells,but a genetic marker has a number of obviousadvantages. It is indelible, heritable and need notdamage a cell or distort its development. However, itis not always easy to introduce a genetic marker into acell, especially if the number of marked cells as wellas the precise time in development at which themarker is introduced need to be controlled.

For several years now, molecular biologists havebeen using a variety of gene transfer techniques (seeGordon & Ruddle, 1985, for review), the mostfamiliar of which to embryologists is probably themicroinjection of DNA into the mouse pronucleus asa means of generating transgenic mice (Gordon et al.1980; Wagner, Stewart & Mintz, 1981; Harbers,Jahner & Jaenisch, 1981; Brinster et al. 1985). How-ever, it is not obvious how the methods of molecularbiology, which are primarily in vitro techniques, canbe 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 representsthe initial studies from a small number of labora-tories, in which retroviruses have been applied to thestudy of cell lineage. In concentrating on this aspectof retroviral technology, I am ignoring several otherinteresting applications of retroviruses (such as in-sertional mutagenesis and gene therapy) and alsoexcluding much of the molecular biology of thestructure and function of retroviruses. (Good reviewsof this area exist in any case: see Varmus, 1982;Coffin, 1985; Bernstein, Berger, Huszar & Dick,1985.)

Retroviruses and retroviral vectors

Retroviruses have a number of properties that makethem versatile and powerful tools in the study ofdevelopment. Their principal advantages stem fromthe fact that they are a naturally evolved system fortransferring genes into cells of a host animal (Fig. 1).As a consequence, this transfer is highly efficient(unlike most artificial means) and also highly accuratein that a faithful copy of the retroviral genome isintegrated into the host cell chromosome. Further-more, the retrovirus itself carries the nucleic acidsequences required to direct the host cell in thetranscription of the retroviral genes. This means thatin many situations the virus can be relied upon toexpress itself without recourse to further geneticmanipulation. On the other hand, viruses can beengineered to provide other promoter elementsshould they be required (Wagner, Vanek & Venn-strom, 1985; Stewart, Vanek & Wagner, 1985; Emer-man & Temin, 1984, 1986).

The structure of the retroviral genome has evolvedwith the retroviral genes located in the middle of thegenome, flanked by sequences called the long ter-minal repeats (LTRs). The significance of the LTRs isthat they contain all the sequences required in cis forthe integration and expression of the retrovirus. Thisorganization is principally a reflection of the mechan-ism of replication that retroviruses have evolved(Varmus, 1982), but it is fortunate from the point ofview of the molecular biologist because it means that,in principle, any gene could be introduced in placeof the retroviral genes, and the virus could still in-fect a cell, integrate and transcribe that cloned gene.(The genetic engineering of retroviruses is essentiallythe same as cloning in plasmids and mostly uses thetechniques of conventional molecular biology.) Suchexpression of cloned DNA in retroviruses has nowbeen shown to work in a number of instances, as willbe described below.

Of equal importance has been the development ofmethods for the packaging of recombinant viruses

410 / . Price

into infectious particles (Fig. 2). This is done bytransfecting a plasmid that contains the engineeredvirus into what is termed a packaging cell line (Mann,Mulligan & Baltimore, 1983; Watanabe & Temin,1983; Cone & Mulligan, 1984; Sorge, Wright, Erd-

Reversetranscription ^ ^ _ D N A

DNA

Fig. 1. The retroviral life cycle. This figure is a schematicdiagram of the life cycle of a typical wild-type retrovirus.The retroviral particle is adsorbed onto the cell plasmamembrane by the binding of its envelope surfaceglycoproteins to a specific surface receptor. Followingfusion, the retroviral genomic RNA passes into thecytoplasm, and is reverse transcribed into DNA, gainsentry into the nucleus and as a proviral circle integratesrandomly into the host cell chromosomal DNA. Theintegrated provirus acts as typical chromosomal DNA inthat it is inherited by both daughter cells whenever thecell divides. The provirus is also transcribed and theretroviral genes are translated, using the cell's normalmachinery. The assembly of a new retroviral particlecompletes the life cycle. The genomic retroviral RNAtranscript comes together with the retroviral geneproducts and buds off to form a new free retroviralparticle.

The above description relates to a wild-type retrovirus.A retroviral vector of the type considered in this review isdeficient in one important aspect of this life cycle. Theretroviral vector is infective in the same manner as awild-type virus, it is reverse transcribed and integrates asnormal, and (depending on the construct) is transcribedand translated. However, because it does not have theretroviral genes, it cannot form the retroviral geneproducts required to assemble new retroviral particles.This is of crucial importance for lineage studies because itmeans that an infected cell cannot pass virus on toneighbouring, uninfected cells. Only its daughter cells willinherit the provirus from an infected cell.

man & Cutting, 1984). This permanent cell linemakes the retroviral gene products but makes no viralgenomic RNA that is capable of forming a retroviralparticle. In other words, it has all the ingredientsnecessary for making a virus with the exception ofpackageable retroviral RNA. The engineered virus,however, once introduced into the cell will form suchan RNA, which consequently is packaged and re-leased by the transfected packaging cells. In thismanner, the tissue culture supernatant from thesecells constitutes a permanent supply of high titrevirus. Viruses of this type, that encode foreign genesbut not the endogenous retroviral genes, are usuallytermed retroviral vectors. They are infective in the

Packaging cell line,produces retroviral proteins

(required for packaging)but no packageable RNA

.0.Engineered

retroviral genomein plasmidTRANSFECTION

neoSELECTION

Clones isolatedand expanded

\ Packaged, engineeredretroviral vector

released by cells intoculture medium

Fig. 2. The production of a retroviral vector using apackaging cell line. When a retroviral vector isconstructed that does not code for the endogenousretroviral genes, some way must be found to package theengineered retroviral genome into a retroviral particle.The most popular method of doing this is to use apackaging cell line. This is a cell line that has beentransfected with a retroviral genome that encodes all theretroviral genes and so makes all the retroviral geneproducts. However, this retroviral genome is deficient ina sequence that is required for packaging. Consequently,the genomic RNA transcribed from this provirus cannotbe packaged even though the retroviral gene products arepresent. One can think of a packaging cell as a cellwaiting to package appropriate RNA into viral particlesbut with no suitable RNA of its own to package.

To package a retroviral vector, the cells are transfectedwith a plasmid that contains an engineered retrovirus.Typically a selectable marker such as the neo gene isincluded in the construct, so that clones of transfectedcells can be selected and isolated. Such clones packagethe engineered virus and release this packaged retrovirusinto the culture medium. Therefore, the cell lines derivedfrom these clones provide a permanent supply of theengineered retrovirus.

Retroviruses and the study of cell lineage 41 ]

same manner as wild-type viruses, but, on infecting acell, they cannot complete the wild-type life cycleshown in Fig. 1, because they do not contain theendogenous retroviral genes required to package anRNA.

A variety of studies has now been done usingretroviral vectors of this type to infect cells in culture.In the earliest experiments, the cloned genes usedwere predominantly those that encode selectablemarker genes such as the Herpes simplex thymidinekinase (tk) gene or the Tn5 neo gene, which infersG418 resistance on eukaryotic cells (see Bernsteinet al. 1985 for references). But more ambitiousconstructs have also begun to appear and the ex-pression of preproparathyroid hormone (HeUermanet al. 1984), granulocyte-macrophage-colony-stimu-lating factor (Lang et al. 1985), the polymeric immu-noglobulin 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 intothe host cell genome, so that the pro virus is inheritedby all the progeny of that cell. Hence, the clone ofcells derived from the infected cell is geneticallymarked. Moreover, when the provirus integrates, itdoes so randomly, so that each integration site isunique. Consequently, on infection each cell (andsubsequent clone) is given a genetic label. So if thehost cell DNA is cut with a restriction enzyme, andthe DNA fragments separated electrophoreticallyand hybridized with a probe recognizing the viralsequences, the fragment of DNA from each clonethat contains the provirus will be unique and of acharacteristic size, which will distinguish it from anyother such fragment.

This manner of marking clones with a retrovirusprovides one way in which they can be used to studycell lineage. One of the advantages of this approach isthat expression of the viral genome is not required,the presence of the integrated provirus being suf-ficient to recognize the clone. However, a potentialdisadvantage of the approach is that in situationswhere there is retroviral expression, infected cells cangenerate new retroviral particles and so spread thevirus in a horizontal fashion, thereby obscuring anyclonal analysis.

The most elegant way of avoiding this problem is touse the retroviral vectors described above, which arereplication defective and, therefore, cannot spreadhorizontally to other cells. I will return to thisapproach to lineage later in this review. However,another solution to the problem is to study systems inwhich virus does not express. For example, the

Muloney murine leukaemia virus (MoMLV) doesnot express in cells of the preimplantation mouse em-bryo (Jaenisch et al. 1975). It appears that the pro-viral DNA becomes methylated (Stewart, Stuhlman,Jahner & Jaenisch, 1982), although it is not clear thatthis is the primary reason for the lack of expression(Gautsch & Wilson, 1983; Niwa, Yokata, Ishida &Sugahara, 1983). Consequently, studies of lineage inthe early mouse embryo are possible using wild-typeMoMLV retrovirus, and these experiments are de-scribed below.

This manner of recognizing clones - studying bandsizes on Southern blots — has been productive in twoprincipal areas of research into cell lineage. These arehaematopoiesis and the early development of themouse embryo.

Haematopoiesis

The development of blood cells was in many ways anobvious place to begin applying gene transfer tech-niques to the study of cell lineage. It is one of a fewvertebrate systems where a considerable amountwas already known about cell lineage relationships,largely as a result of experiments using nonretroviralchromosomal markers (see Quesenberry & Levitt,1979, and Till & McCulloch, 1980 for reviews). Also,techniques already existed for the removal and cul-ture of mouse bone-marrow cells and their sub-sequent introduction into an irradiated syngeneichost. Probably the main drive to research in this areais the hope that the haematopoietic system might beamenable to gene therapy.

The lineage of the haematopoietic system is poss-ibly better understood than that of any other mam-malian system. It has been clear for some time thatthere is a stem cell (CFU-S) that has the ability togenerate the entire myeloid lineage (Till & McCul-loch, 1961). This stem cell is self replicating (Simno-vitch, McCulloch & Till, 1963) and is believed togenerate committed progenitor cells, which can giverise to particular differentiated cell types (Lewis &Trobaugh, 1964; Curry & Trentin, 1967).

Several workers have shown that bone marrowcells can be infected in vitro with retroviral vectorssuch that a proportion of the stem cell populationbecomes marked. On reintroduction into an ir-radiated host, these cells can successfully repopulateboth myeloid and lymphoid cell compartments(Joyner, Keller, Phillips & Bernstein, 1983; Miller etal. 1984; Williams et al. 1984; Hock & Miller, 1986).Although in the earliest studies only a small pro-portion of the stem cells was labelled, techniqueshave more recently been refined so that now it ispossible to label close to 100% of the stem cells(Eglitis, Kantoff, Gilboa & Anderson, 1985; Dick et

412 /. Price

al. 1985; Lemischka, Raulet & Mulligan, 1986). Thisis done generally by coincubating the bone marrowcells with high titre viral producer lines and bypushing the predominantly quiescent stem cells intomitosis with growth factors, or by treating the donoranimal with 5-fluorouracil prior to removing themarrow. This drug kills dividing cells and so pushesstem cells into division to compensate for the lostpopulations. Consequently, there is a higher pro-portion of stem cells in the total bone marrowpopulation. The increased division of the stem cells issignificant because only dividing cells seem able tointegrate virus. The increased proportion of stemcells is significant because normally these cells are atiny minority of the total population and as such arequite difficult to study.

A question of some interest was whether theretroviral technique could be used to label the pluri-potential stem cell that gives rise to both myeloid andlymphoid derivatives. It is now clear from a numberof studies that such cells can be identified with thistechnique (Williams et al. 1984; Dick et al. 1985;Keller, Paige, Gilboa & Wagner, 1985; Lemischka,Raulet & Mulligan, 1986). The importance of thisobservation lies not so much in proving the existenceof a pluripotential haematopoietic stem cell (therehas been good evidence in this regard for many years(Wu, Till, Siminovitch & McColloch, 1968; Abram-son. Miller & Phillips, 1979)) but in demonstratingthe relative ease with which it is possible to label suchcells and follow their fate during development. Withthese better marking techniques, more ambitiousexperiments have become possible and the initialresults are promising. For example, Lemischka andhis colleagues were able to analyse how the contri-bution of marked stem cells to various cell compart-ments varied over periods of months in the sameanimal (Lemischka et al. 1986). Interestingly, theyfound that each cell compartment was made up of thederivatives of relatively few clones, but that over aperiod of 2-3 months the contribution shifted ascertain clones apparently disappeared from the dif-ferentiated cell populations whereas others, pre-viously undetected, now appeared. This is not, ofcourse, a normal animal, recovering as it is fromradiation and bone marrow transplant. Nonetheless,these results suggest that a limited number of stemcells contributes to the haematopoietic system at anyone moment. The system is, in other words, oligo-clonal and, with time, stem cells cease to contributeto the pool of differentiated cells and are replaced.

It was also possible in the same study to analyse thecontribution that marked clones make to variousorgans and anatomical locations in repopulated mice(Lemischka et al. 1986). Consequently, the authorsobserved 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 repopu-lated. A clone could contain, for instance, splenic butnot thymic T cells, or bone marrow but not peritonealmacrophages. (The authors do not, however, de-scribe exactly which combinations of cell types andlocations could be clonally derived and which couldnot.) These data should, of course, be interpretedwith care; just because a stem cell only populatescertain compartments does not prove that its fate wasdetermined and that it could not have populatedother compartments. Nonetheless, this observationraises the possibility that haematopoietic cells canbecome restricted in terms of the anatomical com-partments that they populate as well as in the types ofblood cell to which they give rise. If this conclusionproves to be correct, it will be interesting to see how itinfluences conventional models of haematopoieticcell lineage.

These studies of haematopoiesis are still at an earlystage and they are, I think, clearly going to influencemore than just ideas on lineage. Another obviousdirection is to introduce genes into haematopoieticstem cells with a view to analysing the effect this hason their development. Oncogenes, growth factorsand various surface receptors all come to mind in thisregard, as do the various clinically important genesthat, it is hoped, may become candidates for genetherapy. (See, for example, Ledley, Grenett, McGin-nis-Shelnutt & Woo, 1986; Williams, Orkin & Mulli-gan, 1986; Jolly et al. 1986.)

Lineage analysis in the early mouse embryo

A similar strategy to that described for haematopoi-esis has also been applied to the study of lineage inthe preimplantation mouse embryo. Embryos can beinfected at early stages then introduced into pseudo-pregnant foster mothers for implantation. This ap-proach 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 issimilar to other methods of generating chimaeras butit is notably less invasive, requiring no injection ofDNA or aggregation of embryos. Alternatively, em-bryonic carcinoma (EC) cells (Stewart et al. 1982)or embryo-derived stem cells (ES or EK cells) canbe infected in culture (Evans, Bradley, Kuehn &Robertson, 1985; Robertson, Bradley, Kuehn &Evans, 1986) and then used to form chimaeras usingestablished methods. In this manner, the retroviralsequences are introduced into a subpopulation of theembryonic cells.

These techniques have been used for a number ofpurposes including insertional mutagenesis, which

Retroviruses and the study of cell lineage 413

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 thestudy of lineage, though, this approach has beentaken up by Soriano & Jaenisch (1986) as a means ofanalysing early events in the mouse. They infectedembryos at the 4- to 16-cell stages by cocultivatingthem for 24 h with MoMLV producer cells. Afterintroducing the embryos into a foster mother, theyallowed normal development to proceed and thenwere able to ask in which lineages the progeny ofmarked blastomeres had appeared. For example,they asked whether clones were shared betweenembryonic and extraembryonic tissues, or betweensomatic and germ line; and within the somatic tissues,whether clones were represented in all tissues orrestricted to a subset of organs?

They reached some interesting conclusions. Theyfound very little evidence for a lineage common toboth placenta and embryo at this stage: of 52 proviralintegration sites examined at 14 days post coitum(E14), 25 were found in the embryo, 27 in theplacenta and only 3 in both. In mosaic animals thatwere allowed to progress to adulthood, the authorswere able to assess to what extent a marked blasto-mere that had contributed to one organ, the liver forexample, had also contributed to others. They foundthat in the vast majority of cases, a blastomere thathad contributed to any one organ of the eight or ninethat they analysed, had also contributed to the rest.

They were able to quantify this observation. Bydensitometric scanning of a Southern blot of DNAfrom each tissue, the authors could estimate theintensity of any given proviral band relative to DNAfrom tissues of animals that were heterozygous for theprovirus (that is, all cells of the tissue carried onecopy of the provirus). In other words, if the controlcells were considered to have a value of 1, i.e. onecopy per cell in the entire tissue, then a value of, say,0-5 would mean that half of the cells of that tissue hada copy of that particular provirus (and hence werederived from the blastomere that had been infected).This relative value (which the authors want to call'molarity') gives an estimate of the proportion of thecells in any tissue that are derived from an infectedblastomere. They observed that for any given proviralintegration this value was the same in all tissues. Thatis, the derivatives of marked blastomeres made up aroughly equal proportion of all organs. This was truefor almost all the proviruses analysed (2 exceptionsout of 35). These were, in other words, fine-grainedmosaics. This must mean, as the authors conclude,that considerable stem cell mixing and division mustoccur prior to the setting apart of tissue anlagen.

By breeding chimaeric mice and analysing theappearance of proviruses in the offspring, the authors

were able to show not only that labelled blastomerescontributed to the germ line, but also that some ofthese had failed to contribute to somatic tissues. Thissuggests that at the time of proviral integration, someblastomeres were set aside in the germ cell (asopposed to somatic cell) lineage.

Many of these data fit with those obtained fromchimaeras generated by the aggregation of pre-implantation embryos (Gardner, 1978; Rossant,1984). For example, it has been observed previouslythat blastomeres at such stages can contribute to thesomatic lineage but not to the germ cell line. Theobservation that blastomeres before the 64-cell stagecan contribute to the germ line but not the somaticlineage is slightly more surprising as it implies that thegerm line is set aside at an earlier stage than haspreviously been thought. Certainly, other techniqueshave identified precursors common to both theselineages as late as 5 or 6 days post coitum (McMahon,Fosten & Monk, 1983; Gardner et al. 1985).

As noted above, Soriano and Jaenisch have quanti-fied their Southern blot data by comparing thecontributions made by different clones to a number ofmosaic animals. When all these values were com-pared, the authors found that the lowest value thatappeared was 0-12, even though they estimated thatthey could have detected as low as 0-06. Since a valueof 0-12 is roughly one eighth as a fraction, this meansthat if a clone contributes to a tissue, it contributes atleast an eighth of the cells of that tissue. The authorsinterpret this finding to mean that eight cells are setaside to make the entire embryo.

There are, I think, two immediate problems withthis interpretation. First, one would like some evi-dence that this value of 0-12 has some real signifi-cance. Simply the fact that this is the lowest valuefound in this particular experiment cannot be taken tomean that it is significant biologically without someevidence that it is the invariant minimum. Basically,this is a statistical problem and should be treated assuch. Second, as has already been noted by Rossant(1986), one difficulty with this interpretation, eventaken at face value, is that it is not clear when theseeight cells would be set aside. A problem with thetechnique (as Soriano and Jaenisch point out) is thatalthough infection takes place quite quickly, it seemsby comparison with tissue culture studies that theprovirus can remain in the cell for some time before itactually integrates. Stewart et al. (1982) have esti-mated that with MoMLV infections of F9 cells, thislag can be up to 2 days. This means that although theinfection took place at the 4- to 16-cell stage inSoriano and Jaenisch's experiments, integration (andhence 'marking') may have taken place as late as the64-cell stage. So even if the 8-cell theory is correct,

414 J. Price

one wonders which eight cells are being discussed andwhen are they put aside.

Retroviral vectors encoding histochemicalmarkers

Apart from the intrinsic value of their findings, thestudies of haematopoiesis and embryonic lineagedescribed above have indicated the value of retro-viruses as lineage markers. Furthermore, I think it isfair to say that they have been successful while havingemployed retroviruses to only a fraction of theirpotential. All the lineage studies discussed so far havehad a noticeable limitation. Because the method ofdetection takes the form of a Southern blot, thetechnique is limited to analysing relatively largepopulations of cells. This limitation has two immedi-ate consequences. First, small clones cannot bedetected. Second, it is not known which cells within apopulation belong to the marked clone, so that, if thepopulation contains more than one cell type, it is notpossible to say whether the marked cells includedonly one or many cell types.

Unfortunately, these limitations exclude a numberof the more interesting types of lineage study thatmight be undertaken. For example, in the nervoussystem, it would be interesting to know whetherneurones and glial cells are derived from the same setof progenitor cells, or whether separate glial andneuronal precursors are set aside early in develop-ment. The inability to answer questions of this type isa considerable handicap in developmental biology,not just in the study of neural development. It isparticularly frustrating in trying to move from adescriptive to a mechanistic analysis of developmen-tal events. The reason for this is clear from theexample given above. Unless the timing of differentdecisions and the order in which crucial decisions aremade are known, it is difficult to decide which of thepotential interactions and influences are likely to besignificant. Indeed, much of the renewed interest inlineage in vertebrates stems from the envy with whichthose studying vertebrates look upon the successes inthe invertebrate field. The concept of 'compartments'in insects is well known now and needs no repeatinghere. But it is noteworthy that the understanding oflineage relationships in invertebrates has also beencrucial in unravelling developmental events wherelineage per se plays a minor role in determining fate.One example of this is the development of the insecteye where it has been shown that lineage plays no partin determining cell type (Ready, Hanson & Benzer,1976; Lawrence & Green, 1979). This finding hashelped focus attention on the later cell-cell interac-tions that are apparently the significant events in

determining how cells form an ommatidium (Tomlin-son, 1985; Tomlinson & Ready, 1986; Lebovitz &Ready, 1986).

Fortunately, there are other ways in which aretrovirus can be employed to study lineage. It ispossible to use a vector that not only integrates andgenetically marks a clone, but also expresses aninserted gene. If the product of this gene can beidentified histochemically, then even a single cellexpressing this product can be recognized. This givesthe advantage of being able to study small clones andthe ability to identify precisely which cells within astructure are part of a clone. There are so far twopublished reports of this approach being successfullyapplied. In one of them, Sanes and his collaboratorsat the Pasteur Institute have used a retroviral ex-pression system to look at postimplantation lineagesin the mouse embryo (Sanes, Rubenstein & Nicolas,1986). The other was work in which I have beeninvolved in collaboration with David Turner andConnie Cepko of Harvard Medical School. We haveinvestigated 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 constructedthat expressed the bacterial lacZ gene, which encodesfor the enzyme /3-galactosidase. However, the twoviruses were constructed differently. In our BAGvirus, the lacZ gene comes under the control of theendogenous retroviral promoter and enhancer el-ements, whereas Sanes et al. used an internal SV40early promoter to drive the gene.

/S-galactosidase has a number of obvious advan-tages as a marker. It has a number of convenientassays which make the quantification of expressionstraightforward. Similarly, there are a variety ofmonoclonal and polyclonal antibodies availableagainst the /3-galactosidase protein. Most valuably,there is a quick, sensitive histochemical method todetect cells that are expressing the enzyme. This usesthe substrate X-gal and can be used on cultured cellsor tissue sections or even tissue whole mounts, tostain cells expressing /3-galactosidase.

In addition to these features, it seems likely thatembryonic cells could express high levels of /3-galac-tosidase without it interfering with normal develop-ment. Of course, that is difficult to prove, for suchinterference might be in ways that go unnoticed.However, in other systems, high levels of/3-galactosi-dase expression have occurred with no apparentperturbation (Lis, Simon & Sutton, 1983; Hiromi,Okamoto, Gehring & Hotta, 1986; Goring etal. 1987)and, in the studies considered here, there was noevidence of abnormality (see references below).

Retroviruses and the study of cell lineage 415

Postimplantation lineages

In the study by Sanes et al. (1986), virus was injectedinto the mouse embryo in utero between 7 and 11 dayspost coitum (E7 to El l ) . At this stage of develop-ment, it is not possible to see the embryo properthrough the uterine wall because of the decidualtissue. The only hope is to inject the virus into theamniotic cavity or yolk sac and so expose the embryoto virus. There is also a limit to the amount of virusthat can be injected into such a small structure and itis impressive that these workers could get sufficientvirus into the embryo to infect any cells at all. Despitethis limitation, they were able to study the lineage of anumber of different tissues. In particular, they de-scribe a series of clones in the visceral yolk sac and inthe skin.

When they injected virus at E7, the clones theyfound upon subsequent examination of the yolk saccontained cells from the mesothelial layer, capillaryendoderm and fibroblasts. In other words, all the celltypes in the visceral yolk sac that are mesodermallyderived were found together in clones. Cells of thevisceral endoderm were not labelled. When virus wasinjected at E9, the majority of clones contained onlycapillary endothelial cells and fibroblasts. Only oneclone contained mesothelial cells and it had no othercell type. They also found one clone containingfibroblasts alone. The authors interpret these data tosuggest that at E7 there is a common progenitorwhich gives rise to the three mesodermal derivatives,capillary endothelium, fibroblasts and mesothelium.By E9, however, it seems that progenitors exist thatgive rise to either fibroblasts and capillary endothelialcells, or to mesothelium. This suggests that a commonprogenitor that generates all three cell types gives riseby E9 to two progenitors with a more restrictedpotential.

In the skin, the story is similar. From their E9injections, the authors found clones of epidermal cellsand, in four out of five cases, these clones alsocontained cells of the periderm. (This is the outer-most layer of cells found covering embryonic but notadult skin.) Interestingly, these clones could containcells of both the ordinary epithelium and of the hairgerm, the structure that develops into a hair. Whenthe clones from El l injections were analysed, threeout of four clones contained epidermis but notperiderm and the fourth contained periderm but notepidermis. The conclusion was, therefore, that at E9a bipotential progenitor exists which gives rise to cellsthat by El l can make only epidermis or periderm butnot both. In the yolk sac and skin, therefore, Sanesand his colleagues were able to define a progressiverestriction in the cell types to which ancestral cells

could give rise. They were also able to delineate tosome extent when these restriction events took place.

These studies are important for a number ofreasons. First, they have obviously gone some dis-tance towards sorting out some of the lineage re-lationships of the tissues they have examined. Butalso, this study proves the feasibility of retrovirallineage studies at this stage of development. Whatremains to be seen is whether the lineages of lessaccessible tissues can be studied. One might expect itto be more difficult at, say, E9 to get virus into someof the mesodermal or endodermal embryonic struc-tures as this would require infection of cells withinembryo itself. It remains to be seen whether or notthis is possible.

Lineage studies in the nervous system

The retroviral approach that has been taken tolineage analysis of the nervous system is similar tothat of Sanes etal. (1986) except that we concentratedon the later stages of development in an effort toresolve questions regarding the final generation ofcell diversity (Price et al. 1987; Turner & Cepko,1987). Interestingly, whereas the study of Sanes et al.has indicated lineages that diverge early in develop-ment, the results in the nervous system have shown,somewhat surprisingly, that some types of progenitorcells exist that can give rise to an array of cell typeseven very late in development.

This was shown most clearly in the rat retina. Somecell types of the rat retina are generated embry-onically, but most rod photoreceptors and someamacrine cells, bipolar cells and Miiller glia aregenerated postnatally. If the BAG virus is injectedinto the retinas of new-born rats and their retinasanalysed histochemically in adulthood for clones thatexpress /J-galactosidase, then one finds clones con-taining all these cell types (Price etal. 1987; Turner &Cepko, 1987). It can also be shown that the number ofclones observed is proportional to the amount of virusinjected and that, if the viral titre is calculated in thisfashion, it differs from the in vitro titre on 3T3 cells bya factor of only four (Turner & Cepko, 1987). Theclones were small (two or three cells on average) aswould be expected so late in development, and by farthe majority of clones contained only rods - again asmight be expected given that this is the main cell typebeing generated at this time.

The interesting question is what are the lineagerelationships of these retinal cell types? Turner andCepko have analysed hundreds of clones and not onlydid they find mixed clones containing more than onecell type, but they also observed clones containingevery 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 sizeof clones and the low frequency of occurrence ofsome of the cell types such 'four cell type' cloneswould be expected to be rare.

The simplest interpretation of these data is thateach progenitor in the neonatal rat retina can give riseto all cell types. A less-likely interpretation is thatthere are numerous types of progenitor cell, each ofwhich can give rise to a different combination of thefour cell types. In either case, it seems that a retinalprogenitor cell can give rise to two quite different celltypes up to its final division.

This is very reminiscent of the situation in theinsect retina referred to earlier, but runs somewhatcontrary to what is thought to occur in the mam-malian central nervous system where separate glialand neuronal lineages are thought to be generatedquite early (see Levitt, Cooper & Rakic, 1981; Rakic,1983). This latter view has been supported in recentyears by the work of Raff and his colleagues whichhas shown the existence in the rat optic nerve of abipotential progenitor which can give rise to one typeof astrocyte and oligodendrocyte but not to othertypes of astrocyte or any neuronal cells (Raff, Miller& Noble, 1983). This implies the existence in theoptic nerve, at least, of progenitors that have thepotential to make less than the full complement ofneural cell types.

None of these data are contradictory, of course; thedevelopmental strategy that the animal applies in theretina might be quite different from that used in thebrain. It is now necessary to apply the /?-galactosidaseretroviral marker technique to other regions of theCNS; and that is currently being tried. Sanes et al.(1986) reported finding some clones in the brainalthough no clonal analysis was reported. We haveinfected embryonic cortical cells in culture and,judging primarily by morphological criteria, foundclones of glial cells containing both protoplasmic andfibrous types of astrocytes (Price et al. 1987, andunpublished observations). It is, however, too earlyyet to interpret these results with assurance.

In all of the above discussion, I have ignored onepossible problem with the /3-galactosidase vectorstudies. In the early mouse embryo and haematopoi-etic studies, clonality was recognized by the fact thata proviral integration marked a unique site in thegenome. In the histochemical studies using the/S-galactosidase vectors, this was not possible. Dis-crete areas of stained cells were interpreted as beingclones. There is, however, the possibility that the'clone' was the result of two neighbouring cells beinginfected with virus. In practice, the possibility of twoclones being superimposed is slight as, in most cases,the use of low viral titres ensures that an infection is arare event. For example, Turner and Cepko typically

used titres of virus that gave around one hundredclones (average two cells each) over an entire retina.Similarly, in our experiments with cortical cells invitro (Price et al. 1987) a culture with about 105 cellswould have perhaps five or ten marked clones.Nonetheless, an individual result should be viewedcautiously and, in all the experiments cited here, mosttypes of clones were found several times. The onlyexceptions were some of the clones found by Sanes etal. (1986) in their embryonic injections where, pre-sumably, clones were rare. In the cited studies on theretina, each clone could be positively identified assuch by its strict radial arrangement (an observationof interest in itself) so that even clones quite closetogether could generally be resolved. So clonality isnot a problem unless one believes that there is somepredisposition for infective events to cluster and thereis no evidence for that.

It is likely that the studies described in this revieware just the foretaste of good things to come. Thepotential of retroviruses is only beginning to beexplored; there are many more adventurous retro-viral constructs than those described here. Conse-quently, it should now be possible to establish thelineage relationships of many cells in the vertebrateusing some combination of the techniques reviewedhere. This is an exciting possibility.

I would like to thank Jonathon Cooke, Brigid Hogan,Rob Krumlauf, Andy McMahon, Helen New, Martin Raffand Jim Smith, all of whom were kind enough to read andcomment on this manuscript.

References

ABRAMSON, S., MILLER, R. G. & PHILLIPS, R. A. (1979).The identification in adult bone marrow of pluripotentand restricted stem cells of the myeloid and lymphoidsystems. J. exp. Med. 145, 1567-1579.

BERNSTEIN, A., BERGER, S., HUSZAR, D. & DICK, J.(1985). Gene transfer with retrovirus vectors. InGenetic Engineering, vol. 7 (ed. J. K. Setlow & A.Hollender), pp. 235-261. London: Academic Press.

BRINSTER, R. L., CHEN, H. Y., TRUMBAUER, M. E.,YAGLE, M. L. & PALMITER, R. D. (1985). Factorsaffecting the efficiency of introducing foreign DNA intomice by microinjecting eggs. Proc. natn. Acad. Sci.U.S.A. 82. 4438-4442.

COFFIN, J. (1985). 'Genome structure'. In RNA TumorViruses (ed. R. Weiss, N. Teich, H. Varmus & J.Coffin). New York: Cold Spring Harbor.

CONE, R. D. & MULLIGAN, R. C. (1984). High-efficiencygene transfer into mammalian cells: generation ofhelper-free recombinant retrovirus with broadmammalian host range. Proc. natn. Acad. Sci. U.S.A.81, 6349-6353.

Retroviruses and the study of cell lineage 417

CURRY. J. L. & TRENTIN, J. J. (1967). Hemopoietic spleencolony studies. IV. Phytohemagglutinin andhemopoietic regeneration. J. exp. Med. 126, 819-832.

DEITCHER, D. L., NEUTRA, M. R. & MOSTOV, K. E.

(1986). Functional expression of the polymericimmunoglobulin receptor from cloned cDNA infibroblasts. J. Cell Biol. 102, 911-919.

DICK, J. E.. MAGLI, M. C , HUSZAR, D.. PHILLIPS, R. A.

& BERNSTEIN, A. (1985). Introduction of a selectablegene into primitive stem cells capable of long-termreconstitution of the hematopoietic system of W/Wvmice. Cell 42, 71-79.

EGLITIS, M. A., KANTOFF, P., GILBOA, E. & ANDERSON,

W. F. (1985). Gene expression in mice after highefficiency retroviral-mediated gene transfer. Science230, 1395-1398.

EMERMAN, M. & TEMIN, H. M. (1984). Genes withpromoters in retrovirus vectors can be independentlysuppressed by an epigenetic mechanism. Cell 39,459-467.

EMERMAN, M. & TEMIN, H. M. (1986). Quantitativeanalysis of gene suppression in integrated retrovirusvectors. Molec. cell. Biol. 6, 792-800.

EVANS, M. J.. BRADLEY, A., KUEHN, M. R. &

ROBERTSON, E. J. (1985). The ability of EK cells toform chimeras after selection of clones in G418 andsome observations on the integration of retroviralvector proviral DNA into cells. Cold Spring HarborSymp. quant. Biol. 50, 685-689.

GARDNER, R. L., KELLER, G., LYON, M. F., EVANS, E. P.

& BURTONSHAW, M. D. (1985). Clonal analysis of X-chromosomal inactivation and the origin of the germline in the mouse embryo. /. Embryol. exp. Morph. 88,349-363.

GAUTSCH, J. W. & WILSON, M. C. (1983). Delayed denovo methylation in teratocarcinoma suggestsadditional tissue-specific mechanisms for controllinggene expression. Nature, Lond. 301, 32-37.

GORDON, J. W. & RUDDLE, F. H. (1985). DNA-mediatedgenetic transformation of mouse embryos and bonemarrow - a review. Gene 33, 121-136.

GORDON, J. W., SCANGOS, D. J., PLOTKIN, J. A.,

BARBOSA, J. A. & RUDDLE, F. H. (1980). Genetictransformation of mouse embryos by microinjection ofpurified DNA. Proc. natn. Acad. Sci. U.S.A. 77,7380-7384.

GORING, D. R., ROSSANT, J., CLAPOFF, S., BREITMAN, M.

L. & Tsui, L.-C. (1987). In situ detection of beta-galactosidase in lenses of transgenic mice with agamma-crystallin/lacZ gene. Science 235, 456-458.

HARBERS, K., JAHNER, D. & JAENISCH, R. (1981).Microinjection of cloned retroviral genomes into mousezygotes: integration and expression in the animal.Nature, Lond. 293, 540-542.

HELLERMAN, J. G., CONE, R. C , POTTS, J. T., RICH, A..

MULLIGAN, R. C. & KRONENBERG, H. M. (1984).Secretion of human parathyroid hormone from ratpituitary cells infected with a recombinant retrovirusencoding preproparathyroid hormone. Proc. natn.Acad. Sci. U.S.A. 81, 5340-5344.

HIROMI, Y., OKAMOTO, H., GEHRING. W. J. & HOTTA. Y.

(1986). Germline transformation with Drosophilamutant actin genes induces constitutive expression ofheat shock genes. Cell 44, 293-301.

HOCK, R. A. & MILLER, A. D. (1986). Retrovirus-mediated transfer and expression of drug resistancegenes in human haematopoietic progenitor cells.Nature, Lond. 320, 275-277.

JAENISCH, R., BREINDL, M., HARBERS, K., JAHNER, D. &

LOHLER, J. (1985). Retroviruses and insertionalmutagenesis. Cold Spring Harbor Svmp. quant. Biol. 50,439-445.

JAENISCH, R., FAN, H. & CROKER, B. (1975). Infection ofpreimplantation mouse embryos and of newborn micewith leukemia virus: tissue distribution of viral DNAand RNA and leukemogenesis in the adult animal.Proc. natn. Acad. Sci. U.S.A. 72, 4008-4012.

JOLLY, D. J., WILLIS, R. C. & FRIEDMANN, T. (1986).

Variable stability of a selectable provirus afterretroviral vector gene transfer into human cells. Molec.cell. Biol. 6, 1141-1147.

JOYNER, A., KELLER, G., PHILLIPS, R. A. & BERNSTEIN,

A. (1983). Retrovirus transfer of a bacterial gene intomouse haematopoietic progenitor cells. Nature, Lond.305, 556-558.

KELLER, G., PAIGE, C , GILBOA, E. & WAGNER, E. F.

(1985). Expression of a foreign gene in meyloid andlymphoid cells derived from multipotentialhaematopoietic precursors. Nature, Lond. 318, 149-154.

KJNG, W., PATEL, M. D., LOBEL, L. I., GOFF, S. P. &

NGUYEN-HUU, M. C. (1985). Insertional mutagenesis ofembryonal carcinoma cells by retroviruses. Science 228,554-558.

LANG, R. A., METCALF, D., GOUGH, N. M., DUNN, A. R.

& GONDA, T. J. (1985). Expression of a hemopoieticgrowth factor cDNA in a factor-dependent cell lineresults in autonomous growth and tumorigenicity. Cell43, 531-542.

LAWRENCE, P. A. & GREEN, S. M. (1979). Cell lineage inthe developing retina of Drosophila. Devi Biol. 71,142-152.

LEBOVITZ, R. M. & READY, D. F. (1986). Ommatidialdevelopment in Drosophila eye disc fragments. DeviBiol. 117, 663-671.

LEDLEY, F. D., GRENETT, H. E., MCGINNIS-SHELNUTT, M.

& Woo, S. L. C. (1986). Retroviral-mediated genetransfer of human phenylalanine hydroxylase into NIH3T3 and hepatoma cells. Proc. natn. Acad. Sci. U.S.A.83, 409-413.

LEMISCHKA, I. R., RAULET, D. H. & MULLIGAN, R. C.

(1986). Developmental potential and dynamicbehaviour of hematopoietic stem cells. Cell 45,917-927.

LEVITT, P., COOPER, M. L. & RAKIC, P. (1981).

Coexistence of neuronal and glial precursor cells in thecerebral ventricular zone of the fetal monkey: andultrastructural immunoperoxidase analysis. J. Neurosci.1, 27-39.

LEWIS, J. P. & TROBAUGH, F. E. (1964). Haematopoieticstem cells. Nature, Lond. 204, 589-590.

418 J. Price

Lis, J. T., SIMON, J. A. & SUTTON, C. A. (1983). New

heat shock puffs and B-galactosidase activity resultingfrom transformation of drosophila with an hsp70-lacZhybrid gene. Cell 35, 403-410.

MANN, R., MULLIGAN, R. C. & BALTIMORE, D. (1983).Construction of a retrovirus packaging mutant and itsuse to produce helper-free defective retrovirus. Cell 33.153-159.

MCMAHON, A., FOSTEN, M. & MONK, M. (1983). X-chromosome inactivation mosaicism in the three germlayers and the germ line of the mouse embryo. J.Embryol. exp. Morph. 74, 207-220.

MILLER, A. D., ECKNER, R. J., JOLLY, D. J., FRIEDMAN,

T. & VERMA, I. M. (1984). Expression of a retrovirus

encoding human HPRT in mice. Science 225, 631-632.NlWA, O . , YOKATA, Y. , ISHIDA, H . & SUGAHARA, T.

(1983). Independent mechanisms involved insuppression of the Moloney Leukemia virus genomeduring differentiation of murine teratocarcinoma cells.Ce//32, 1105-1113.

PRICE, J., TURNER, D. & CEPKO, C. (1987). Lineageanalysis in the vertebrate nervous system by retrovirus-mediated gene transfer. Proc. natn. Acad. Sci. U.S.A.84. 156-160.

QUESENBERRY, P. & LEVITT, L. (1979). Hematopoieticstem cells. New Eng. J. Med. 301, 755-766, 819-823and 868-872.

RAFF, M. C , MILLER, R. H. & NOBLE, M. (1983). A glialprogenitor cell that develops in vitro into an astrocyteor an oligodendrocyte depending on the culturemedium. Nature, Lond. 303, 390-3%.

RAKIC, P. (1983). Emergence of neuronal and glial celllineages in primate brain. In Cellular and MolecularBiology of Neuronal Development (ed. 1. Black), pp.29-50. New York: Plenum.

READY. D. F., HANSON, T. E. & BENZER, S. (1976).

Development of the Drosophila retina, aneurocrystalline lattice. Devi Biol. 53, 217-240.

ROBERTSON, E. J. (1986). Pluripotential stem cell lines asa route into the mouse germ line. Trends Genet. 2,9-13.

ROBERTSON, E., BRADLEY, A., KUEHN, M. & EVANS, M.

(1986). Germ-line transmission of genes introduced intocultured pluripotential cells by retroviral vectors.Nature, Lond. 323, 445-448.

ROSSANT, J. (1986). Retroviral mosaics: a new approachto cell lineage analysis in the mouse embryo. TrendsGenet. 2, 302-303.

RUBENSTEIN, J. L. R., NICOLAS, J.-F. & JACOB, F. (1986).Introduction of genes into preimplantation mouseembryos by use of a defective recombinant retrovirus.Proc. natn. Acad. Sci. U.S.A. 83, 366-368.

SANES, J. R., RUBENSTEIN, J. L. R. & NICOLAS. J.-F.

(1986). Use of a recombinant retrovirus to study post-implantation cell lineage in mouse embryos. EMBO J.5. 3133-3142.

SCHNIEKE, A., HABERS, K. & JAENISCH, R. (1983).Embryonic lethal mutation in mice induced byretrovirus insertion into the al(I) collagen gene.Nature, Lond. 304. 315-320.

SCHWARZBAUER, J. E . , MULLIGAN, R. C. & HYNES, R. O.

(1987). Efficient and stable expression of recombinantfibronectin polypeptides. Proc. natn. Acad. Sci. U.S.A.84, 754-758.

SIMNOVTTCH, L., MCCULLOCH, E. A. & TILL, J. E. (1963).

The distribution of colony forming cells among spleencolonies. J. cell. comp. Physiol. 62, 327-336.

SORGE, J., WRIGHT, D., ERDMAN, V. D. & CUTTING, A.

E. (1984). Amphotropic retrovirus vector system forhuman cell gene transfer. Molec. cell. Biol. 4,1730-1737.

SORIANO, P. & JAENISCH, R. (1986). Retroviruses asprobes for mammalian development: allocation of cellsto the somatic and germ cell lineages. Cell 46, 19-29.

STEWART, C. L., STUHLMANN, H., JAHNER, D. &

JAENISCH, R. (1982). De novo methylation, expression,and infectivity of retroviral genomes introduced intoembryonal carcinoma cells. Proc. natn. Acad. Sci.U.S.A. 79, 4098-4102.

STEWART, C. L., SCHUETZE, S., VANEK, M. & WAGNER.

E. F. (1987). Expression of retroviral vectors intransgenic mice obtained by embryo infection. EMBOJ. 6, 383-388.

STEWART, C. L., VANEK, M. & WAGNER, E. F. (1985).Expression of foreign genes from retroviral vectors inmouse teratocarcinoma chimeras. EMBO J. 4,3701-3709.

TILL, J. E. & MCCULLOCH, E. A. (1961). A directmeasurement of the radiation sensitivity of normalmouse bone marrow cells. Radiat. Res. 14, 213-222.

TILL, J. E. & MCCULLOCH, E. A. (1980). Hemopoieticstem cell differentiation. Biochim. Biophys. Ada 605,431-459.

TOMLINSON, A. (1985). The cellular dynamics of patternformation in the eye of Drosophila. J. Embryol. exp.Morph. 89, 313-331.

TOMUNSON, A. & READY, D. F. (1986). Sevenless: a cell-specific homeotic mutation of the Drosophila eye.Science 231, 400-402.

TURNER, D. & CEPKO, C. (1987). Cell lineage in the ratretina: a common progenitor for neurons and gliapersists late in development. Nature, Lond. 328,131-136.

VARMUS, H. E. (1982). Form and function of retroviralproviruses. Science 216, 812-820.

WAGNER, E. F., STEWART, T. A. & MINTZ, B. (1981). Thehuman B-globin gene and a functional viral thymidinekinase gene in developing mice. Proc. natn. Acad. Sci.U.S.A. 79, 5016-5020.

WAGNER, E. F., VANEK, M. & VENNSTROM, B. (1985).

Transfer of genes into embryonal carcinoma cells byretrovirus infection: efficient expression from aninternal promoter. EMBO J. 4, 663-666.

WATANABE, S. & TEMIN, H. M. (1983). Construction of ahelper cell line for avian reticuloendotheliosis viruscloning vectors. Molec. cell. Biol. 3, 2241-2249.

WILLIAMS. D. A.. LEMISCHKA, I. R., NATHAN, D. G. &

MULLIGAN, R. C. (1984). Introduction of new geneticmaterial into pluripotent haematopoietic stem cells ofthe mouse. Nature, Lond. 310, 476-481.

Retroviruses and the study of cell lineage 419

WILLIAMS, D. A., ORKIN, S. H. & MULLIGAN, R. C. WU, A. M., TILL, J. E., SIMINOVITCH, L. & MCCULLOCH.(1986). Retrovirus-mediated transfer of human E. A. (1968). Cytological evidence for a relationshipadenosine deaminase gene sequences into cells in between normal hematopoietic colony-forming cellsculture and into murine hematopoietic cells in vivo. and cells of the immune system. J. exp. Med. 127.Proc. natn. Acad. Sci. U.S.A. 83, 2566-2570. 455-463.