Download Retrovi ruses and the study of cell lineage

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