1 Embryology Unit, Children's Medical Research Institute, University of Sydney,
Westmead, New South Wales, Australia
2 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario,
Canada
e-mail: ptam{at}cmri.usyd.edu.au and rossant{at}mshri.on.ca
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SUMMARY |
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Introduction |
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The making of chimeras by injecting cells into blastocysts, devised by
Richard Gardner and Ralph Brinster, opened up new possibilities for
introducing foreign cells into the embryo. Pluripotent embryonic stem (ES)
cells derived from the inner cell mass of the blastocyst
(Fig. 1), can differentiate
into all tissue types in chimeras, including the germline
(Robertson, 1986). The
demonstration by Oliver Smithies and Mario Capecchi that genes could be
mutated by homologous recombination in ES cells
(Doetschman et al., 1988
;
Thomas and Capecchi, 1989) ushered in a new era of targeted mutagenesis in the
mouse. Chimera production using altered ES cells became a key tool for
generating designer mice. However, chimeras are more than just a tool for
making mouse mutants; they are crucial for analyzing the biological effects of
genetic changes. It is this use we will discuss here, after describing the
different approaches to generating chimeras and the types of markers used to
distinguish the mixed population of cells.
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The building blocks for chimera production |
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Chimeras can be made by combining two whole eight-cell embryos
(Fig. 2A, part I) or by
combining subsets of blastomeres of two cleavage (two- to eight-cell) stage
embryos. Because, at these stages, the early embryonic cells are not yet
restricted in their lineage potency to contribute to the inner cell mass or
the trophectoderm, they are equally capable of contributing to both lineages.
When two diploid eight-cell embryos or blastomeres of two diploid embryos are
aggregated together, chimerism can occur in the epiblast, the primitive
endoderm and trophectoderm (Fig.
2A, parts II,III; Table
1). By contrast, when the inner cell mass (ICM) cells of a diploid
blastocyst are used to make the chimera, whether injected microsurgically into
morula or blastocyst, or aggregated with eight-cell diploid embryos, they
contribute only to the epiblast and to the primitive endoderm, and not to the
trophectoderm because of more restricted lineage potency of the ICM cells
(Table 1). ES cells in the same
situations behave more like epiblast cells
(Table 1). They contribute only
to germ layers that give rise to all the embryonic tissues and some
extra-embryonic tissues (including the amnion, the mesoderm of the yolk sac,
the allantois and the embryo-derived blood vessels in the placenta)
(Beddington and Robertson,
1989) (Fig. 2B, parts II,III; Table 1), but not
to trophectoderm or primitive endoderm, despite their ability to differentiate
into the latter cell type in vitro. Trophoblast stem (TS) cells, which are
permanent cell lines derived either from the trophectoderm of the blastocyst
or from early postimplantation trophoblasts
(Tanaka et al., 1998
),
contribute only to trophectoderm derivatives of the chimeras following
injection into the blastocyst (Fig.
2D, parts II,III; Table
1).
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Lineage markers for chimera analysis |
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Making chimeras of different tissue constitutions |
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ES celldiploid embryo chimeras
Currently, the most common tool for studying mutant phenotypes is the ES
cellembryo chimera, which is generated by introducing wild-type ES cells
into mutant embryos or vice versa. In both cases, the genetic constitution of
the epiblast derivatives (the mouse itself) will be a mixture of mutant and
wild-type cells. However, ES cells do not contribute to the primitive endoderm
or trophectoderm, so the composition of the extra-embryonic primitive endoderm
and trophectoderm will differ depending on the genotype of the ES cells and
embryo used. In a wild-type ES cell
mutant embryo chimera, the primitive
endoderm and trophectoderm will be mutant; whereas in a mutant ES
cell
wild-type embryo chimera, the primitive endoderm and trophectoderm
will be wild type. ES cell
embryo chimeras can be used to distinguish
between the embryonic and extra-embryonic effects of genetic mutations, as
well as to determine lineage-specific effects of mutations in the embryo
proper. If homozygous mutant ES cells are used, the extra-embryonic tissues
will not contain the mutation, all chimeras will be informative and no
genotyping will be necessary. However, if wild-type ES cells are used with
mutant embryos, the extra-embryonic tissues, as well as some tissues of the
embryo, may contain the mutation. Hence, interpreting the impact of mutation
on the development of the chimera will require knowledge of the genotype of
the embryo into which ES cells are introduced. This is accomplished by
sampling extra-embryonic tissues (either visceral endoderm or trophoblasts) of
the chimera to determine which chimeras contain cells from the heterozygous or
homozygous mutant embryos. In contrast to the situation of the
embryo
embryo chimera, genotyping using the extra-embryonic tissue of a
wild-type ES cell
mutant embryo chimera is not complicated by the
presence of wild-type cells originating from the ES cells. A simpler assay for
the presence of the wild-type and a mutant allele will suffice to distinguish
all possible genotypes.
ES cell'tetraploid embryo chimeras
In an ES celltetraploid embryo chimera, the ES cells contribute
primarily to the epiblast-derived tissues, whereas cells of the tetraploid
embryo mainly give rise to the extra-embryonic primitive endoderm and
trophectoderm. The almost complete segregation of descendants of the ES and
tetraploid cells provides a powerful means for revealing the effect of the
mutation on the embryonic versus the extra-embryonic tissues (Box 1). In
addition, as the fetus proper is constituted exclusively by the ES cells in
this type of chimeras, ES cell-derived embryos and adult mice of the same
genotype as the ES cells can be produced immediately for phenotypic studies
(Box 1). Large numbers of embryos of the genotype of the ES cells can be made
by this means, bypassing the tedious process of breeding mutant mice. Finally,
ES cell
tetraploid embryo chimeras may provide an innovative means for
high-throughput analysis of gene function. In addition to the conventional
genomic modification by transgenesis and gene targeting in the ES cells, the
function of single or multiple genes could be altered efficiently by knocking
down the transcriptional activities in the ES cells using RNAi reagents. Using
the ES cell
tetraploid embryo chimeras as the experimental tool, a
substantial number of ES cells can be screened efficiently for the phenotypic
effects of the RNAi treatment (Kunath et
al., 2003
).
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Application of chimera analysis in dissecting complex phenotypes |
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Goal 1: determining lineage-specific gene function
Chimeras that contain normal and mutant cells have proven to be highly
effective for determining the function of specific genes essential for the
specification or differentiation of certain tissue lineages. This is because
the impact of gene function on a specific lineage can be revealed by the
abnormal differentiation of mutant cells from the lineage in question or, in
extreme cases, by a reduced contribution to, or the exclusion of mutant cells
from, specific types of tissues in the chimera.
In an example of this, loss of function of a multitude of genes, such as
Madh2 (previously Smad2), Tcf2 (previously
Hnf1), Foxh1, ß-catenin, Mixl1 and
Sox17, results in different phenotypes of the mutant embryo, ranging
from arrested gastrulation to abnormal formation of the head or trunk, which
superficially have little in common and would not have revealed any specific
function in endoderm development. However, in ES cellembryo chimeras, ES
cells that lack the function of any one of these genes are consistently
under-represented in or excluded from the gut endoderm of the embryo
(Coffinier et al., 1999
;
Hart et al., 2002
;
Hoodless et al., 2001
;
Kanai-Azuma et al., 2002
;
Lickert et al., 2002
;
Tremblay et al., 2001
;
Yamamoto et al., 2001
). The
impact of these mutations on the endoderm potency of the ES cells therefore
reveals the heretofore unrecognized requirement of the function of these genes
for endoderm specification, differentiation and maintenance.
An innovative approach was recently undertaken to test the role of
Egr2 (previously Krox20) in the sorting properties of cells
in the rhombomeres of the embryonic hindbrain
(Voiculescu et al., 2001). In
this approach, the chimera analysis was performed by aggregating wild-type and
Egr2lacZ/Cre R26R embryos. R26R is a Cre-dependent
lacZ reporter transgene. In ES cells derived from
Egr2lacZ/Cre R26R embryos, expression of Egr2 from both
Egr2 alleles is lost. This is due to the targeted mutations caused by
the integration of the lacZ and the Cre-recombinase genes. When the
Egr2 gene is activated in the descendants of these cells in the
hindbrain, the cells will express the lacZ and the Cre
transgene, but only as far as the Egr2 locus remains active. However,
the transient expression of Cre recombinase in the
Egr2lacZ/Cre cells will permanently activate the
lacZ reporter of the R26R locus. This allows the fates of
the Egr2-null cells to be tracked by the R26R lacZ
expression for an extended period of development long after the expression of
the Egr2lacZ allele has ceased. Results of this study
showed that Egr2-null cells fail to mix normally with wild-type cells
in rhombomere 5, but are able to mix with the adjacent even-number
rhombomeres, demonstrating compellingly that Egr2 function is
essential for the maintenance of segment identity
(Voiculescu et al., 2001
).
Goal 2: dissecting gene functions in extra-embryonic versus embryonic
tissues for embryonic patterning
Embryological and gene expression studies have shown that the early
patterning of the anteroposterior axis of the mouse embryo at around the time
of gastrulation requires signalling and transcriptional activity in both the
extra-embryonic and embryonic tissues
(Beddington and Robertson,
1999). The analysis of mutations in candidate patterning genes in
chimeric mice has played a key role in elucidating the importance of
extra-embryonic tissues as sources of patterning signals in the early mouse
embryo. ES cell
diploid chimeras or, more commonly, ES
cell
tetraploid chimeras can be used to distinguish whether a patterning
defect is caused by the embryonic or extra-embryonic effects of a
mutation.
Three possible outcomes may be obtained from these ES celldiploid and
ES cell
tetraploid chimera experiments.
The first possible outcome is that the loss of gene function in the
extra-embryonic tissues alone results in a phenocopy of (i.e. similar
phenotype to) the null mutation and wild-type extra-embryonic tissues can
rescue this mutant phenotype. For example,
Hnf4/ embryos fail to undergo gastrulation,
and have an abnormal epiblast. However, the null phenotype is rescued by
wild-type primitive endoderm and trophectoderm in
Hnf4/ ES celltetraploid chimeras
(Duncan et al., 1997
). This
outcome indicates that Hnf4 function in the extra-embryonic tissues
is crucial for normal gastrulation. Similarly, a null mutation of the
Tcfap2c gene that encodes the AP2
factor is embryonic lethal,
but the presence of wild-type extra-embryonic tissues allows mutant embryos to
survive. By contrast, wild-type ES cells could not rescue the lethality of
Tcfap2c/ embryos because of the defective
extra-embryonic tissues (Auman et al.,
2002
). Defects in early embryonic patterning that are associated
with the abnormal function of the primitive streak (such as those caused by
mutations of Amn or Nodal), or with the abnormal function of
the gastrula organizer (such as those caused by mutations of Akd or
Foxa2), can be partially rescued by the presence of wild-type
extra-embryonic tissues. Therefore, these studies showed that Amn, Nodal,
Akd and Foxa2 function is essential in the extra-embryonic
tissues for normal embryogenesis and that early patterning events must involve
a complex interplay between embryonic and extra-embryonic tissues
(Dufort et al., 1998
;
Episkopou et al., 2001
;
Kalantry et al., 2001
;
Varlet et al., 1997
).
In these chimeras, the extra-embryonic tissues consist of cells of two
different lineages: the trophectoderm and the primitive endoderm. Thus,
without knowing the cell types in which the gene of interest is expressed, it
is not possible to conclude from such experiments whether gene function is
specifically required in the derivatives of the trophectoderm or the primitive
endoderm or in subsets of cells in both tissues. The following example,
however, shows that this ambiguous aspect of chimera analysis may be overcome
if it is known which tissue expresses the gene of interest.
Otx2/ embryos display abnormal development
of the fore- and midbrain. In Otx2/ ES
celldiploid chimeras, the anterior neural plate initially develops
normally, even though over 90% of the embryo consists of
Otx2/ cells, suggesting that normal Otx2
function in the extra-embryonic tissue is sufficient and essential for the for
early morphogenesis of the anterior neural primordium. As Otx2 is not
expressed in the trophectoderm derivatives, its function is therefore likely
to be required only in the visceral endoderm
(Rhinn et al., 1998
).
The second possible outcome is that the loss of gene function in the
extra-embryonic tissues does not cause the mutant phenotype. For example,
Dkk1, Lhx1 (previously Lim1), Hhex (previously
Hex) and Hesx1 are expressed in the visceral endoderm of the
mouse embryo prior to and at early gastrulation and later in the mesoderm or
the ectoderm of the embryo
(Martinez-Barbera et al.,
2000a; Martinez-Barbera et
al., 2000b
; Mukhopadhyay et
al., 2001
; Shawlot et al.,
1999
). Chimeras that contain mutant extra-embryonic tissues
harboring mutations in one of these genes display normal gastrulation, even
when a mixture of wild-type and mutant cells is present in the embryo. By
contrast, chimeras that contain wild-type extra-embryonic tissues and mutant
embryonic cells form abnormal head structures like the null mutant
(Martinez-Barbera et al.,
2000a
; Martinez-Barbera et
al., 2000b
; Mukhopadhyay et
al., 2001
; Shawlot et al.,
1999
). These findings indicate that the function of these genes is
required in the embryonic tissues and not the extra-embryonic tissues.
In the third scenario, loss of gene function in either embryonic or
extra-embryonic tissues results in a mutant phenotype. This suggests that the
function of a mutated gene is essential for both tissue types. For example, in
chimeras that lack Bmp4 activity in the extra-embryonic tissue,
primordial germ cells are not formed from the proximal epiblast. In
Bmp4-null ES celltetraploid chimeras, the lack of Bmp4
activity in the ES cell-derived extra-embryonic mesoderm does not affect germ
cell formation but does disrupt the localization of the germ cells, the
formation of the allantois and the establishment of left-right asymmetry
(Fujiwara et al., 2001
;
Fujiwara et al., 2002
).
Sox2 is another example of a gene that is required in both embryonic
and extra-embryonic tissues. Chimeric analysis of Sox2 function has
revealed that the viability of the epiblast in
Sox2/ mutant embryos can be restored by
wild-type ES cells. However, such chimeras fail to survive beyond
gastrulation, even when the embryo proper consists of predominantly wild-type
ES cells. Chimeras formed by aggregating
Sox2/ ES cells with tetraploid embryos,
however, survive for much longer, suggesting that the failure of Sox2 mutant
embryos to develop beyond gastrulation is because of an extra-embryonic,
rather than an embryonic, defect (Avilion
et al., 2003
). These chimera studies show that Bmp4 and
Sox2 functions are required in both extra-embryonic and embryonic
tissues to sustain development.
Goal 3: identifying cell-autonomous and non-cell-autonomous gene
function
The effect of the mutation may affect only the cells that are expressing
the gene and not other genotypically mutant cells in the same animal. The
restriction of phenotypic effects reflects the cell-autonomous requirement of
gene function. Alternatively, a mutant phenotype could arise by a mutation
that impacts not only on the cells expressing the genetic activity but also on
other cells that do not express the gene. The non-cell-autonomous action of
the gene will mean that the normal function of the gene is not restricted to
any cell population. Analysis of phenotype in an embryo containing only mutant
cells is therefore insufficient for distinguishing between these two modes of
gene action. However, chimera analysis can reveal whether the gene of interest
functions in a cell-autonomous or non-cell-autonomous manner.
In the chimera, cell-autonomous gene function may be revealed by the
exclusion of mutant cells from a certain tissue lineage or by the expression
of an abnormal phenotype in only those cells with the mutant genotype. For
example, in a chimera that lacks either Fgfr1 or T function,
cells fail to ingress properly through the primitive streak, resulting in the
accumulation of the mutant cells in the posterior region of the chimera
(Ciruna et al., 1997;
Wilson et al., 1995
). These
defects in cell movement persist in the presence of wild-type cells in the
chimera, suggesting that each of these genes function in a cell-autonomous
manner. An example of non-cell-autonomous gene action is that of
Foxd3. Loss of Foxd3 function results in a poorly formed
epiblast, an absent primitive streak, abnormal extra-embryonic endoderm and
the demise of embryos shortly after gastrulation. The presence of a small
number of wild-type cells in the embryo proper of a
Foxd3/ ES cell
diploid chimera rescues
these developmental defects. Foxd3 therefore seems to act in a
non-cell-autonomous manner, presumably by regulating cell-cell signalling
activity (Hanna et al.,
2002
).
Some genes act in either cell-autonomous or cell-non-autonomous mode in
different tissue lineages. Ascl2/
(previously Mash2) mutant embryos die early in development because of
a placental deficiency that is associated with a lack of spongiotrophoblasts
and labyrinthine trophoblasts. In the chimeric placenta,
Ascl2-deficient cells are excluded from the spongiotrophoblasts but
not the labyrinthine trophoblasts, suggesting that Ascl2 activity is
required cell-autonomously in the formation of the spongiotrophoblasts but
non-cell-autonomously for that of the labyrinthine trophoblasts
(Tanaka et al., 1997).
Similarly, Eed function is required in a non-cell-autonomous manner
as Eed/ cells can participate in
gastrulation when wild-type cells are present, but not in their absence.
However, Eed is required autonomously in cells to enable their
differentiation to forebrain tissues and somites, as
Eed/ cells are excluded from these tissues
of the chimera. Tetraploid rescue experiments have shown that the defective
morphogenesis of Eed null embryos cannot be rescued by wild-type
extra-embryonic tissues because Eed acts cell-autonomously in the
extra-embryonic tissues (Morin-Kensicki et
al., 2001
).
Goal 4: distinguishing between primary versus secondary defects
The mutant phenotype is a culmination of the loss of gene function in the
primary target cells (the primary effect), as well as additional effects that
are elicited in other tissues by changes in the function of the primary target
cells (the secondary effect). In chimeras, the primary effects of a mutation
can be distinguished from the secondary effects by the ability to tightly
associate a phenotype with the preponderance of mutant cells in the primary
target tissues.
Using ES celldiploid embryo chimeras, it has been possible to show
that the definitive (gut) endoderm is the primary target tissue of
Mixl1 and Sox17 function. In chimeras with extensive
Mixl1 or Sox17 mutant contributions, only the gut is
populated by wild-type cells. Another significant feature is that the
development of the head (of the Mixl1/ ES
cell
embryo chimera) and of the trunk (of the
Sox17/ ES cell
embryo chimera) is
significantly more advanced than in embryos null for either gene. These
results indicate that, in addition to the primary function of these two genes
in endoderm formation, they also have a secondary role in the provision of
morphogenetic activity by the gut endoderm for the development of specific
body parts (Hart et al., 2002
;
Kanai-Azuma et al., 2002
).
Loss of the function of the tumor suppressor retinoblastoma (Rb)
gene in the mouse embryo leads to neural and erythroid tissue defects,
neuronal apoptosis and early embryonic lethality. However, chimeras produced
by the aggregation of Rb/ ES cells with
tetraploid wild type embryos are viable and free from neural and blood
abnormalities, suggesting that these features of the null phenotype are caused
secondarily by extra-embryonic dysfunction, most likely of the placenta.
Nevertheless, rescued mice still have abnormal lens development and show
elevated cell proliferation in the nervous tissues, which are typical of
Rb mutant mice, implying that these may occur as a result of the loss
of Rb in these tissues, an effect that is unrelated to the placental
deficiency (de Bruin et al.,
2003; Wu et al.,
2003
). Secondary defects in an embryo that arise as a result of
placental dysfunction are fairly common. Chimera analysis should thus be a
standard part of phenotypic investigation of mutant embryos in cases of
midgestation lethality, even where the embryonic defects appear to be fairly
specific.
Goal 5: expediting phenotypic analysis
The early death of a mutant embryo is a major factor that can confound the
assessment of all later aspects of gene function. In chimeras, the
incorporation of wild-type cells into the embryo or the extra-embryonic
tissues can rescue the embryo from this early lethality. If gene function
becomes crucial later in development, the resultant chimeric embryo may
develop an abnormal phenotype that is not found in the null mutant embryo. For
example, chimeras containing PLC-deficient cells are viable due to the
restoration of the hematopoietic function by wild-type cells. However, the
development of renal abnormalities in the mutant embryo has revealed an
otherwise undetected cell-autonomous function of PLC
in kidney
formation (Shirane et al.,
2001
). Subtle variations in the requirement of gene function by
cells in different parts of an organ can also be revealed by chimera analysis.
For example, in mutant ES cell
diploid chimeras with an overwhelming
presence of the mutant cells in the embryo proper,
Sox17/,
Mixl1/ and
Madh2/ cells are excluded from the mid- and
hindgut but, conversely, Foxa2/ and
Foxh1/ cells are excluded from the fore- and
midgut (Hart et al., 2002
;
Hoodless et al., 2001
;
Kanai-Azuma et al., 2002
;
Tremblay et al., 2000
;
Yamamoto et al., 2001
). The
loss of the potency (hence the exclusion) of the mutant cells to populate
specific segments of the embryonic gut implies that the endoderm in different
gut segments requires the activity of different genes for its formation or
maintenance
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Future perspectives |
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ACKNOWLEDGMENTS |
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