/. Embryo!, exp. Morph. Vol. 33, 2, pp. 447-457, 1975
Printed in Great Britain
447
Marker chromosome analysis of two mouse
chimaeras
By C. E. FORD, 1 E. P. EVANS 1 AND R. L. GARDNER 2
From the Sir William Dunn School of Pathology and the
Department of Zoology, University of Oxford
SUMMARY
Chimaeric mice were obtained by injecting embryonic (CBA/H-T6 x PT>E)F1 cells into
PDE blastocysts. Three of 15 young were overt chimaeras. One female and one male chimaera
survived to adulthood and after completion of test breeding, which demonstrated chimaerism
in the germ cells of both, they were killed for study when aged 32 and 33 weeks respectively.
Chromosome spreads were scored for the presence or absence of the T6 marker chromosome
in direct preparations from bone marrow, spleen, thymus, lymph nodes, Peyer's patches,
corneas, gut epithelium and testes. Preparations from monolayer cultures of skin, kidney,
ovaiy and gut and from mitogen-stimulated blood cultures were scored in the same way.
Both components of the chimaeras were identified in every one of 53 specimens studied,
some of which, such as single lymph nodes, corneas and segments of gut, may not have
contained more than 105 proliferating cells. This result complements published evidence for
fine-grained mixture obtained in morula-aggregation chimaeras by other methods and implies
extensive cell movement during embryogenesis.
Results obtained from the lymphomyeloid tissues show a clear partition into two groups
in respect of the proportions of host-type to donor-type cells identified. The one group consists of bone marrow, thymus and Peyer's patches, the other of spleen and lymph nodes.
This result would be most simply explained in terms of two distinct stem cell pools and
appears to conflict with the currently favoured hypothesis of a single stem cell pool for the
whole lymphomyeloid complex located in bone marrow. Four groups of factors may, however, modify the relative representation of the two components in different lymphomyeloid
sites: (1) The magnitude of embryonic founder populations. (2) Limited recruitment from the
stem cell pool in post-natal life. (3) Variable size of clones produced by individual stem cells.
(4) Differential cellular behaviour determined by genotypic differences.
INTRODUCTION
Since the introduction of the morula aggregation method by Tarkowski
(1961) and Mintz (1962#), mouse chimaeras obtained by manipulation of preimplantation embryos have become of great experimental interest. The small
T6 translocation chromosome (Ford, Hamerton, Barnes & Loutit, 1956) has
been used as a cell marker to estimate the degree of chimaerism in germ cells
1
Authors' address: Medical Research Council's External Staff, Sir William Dunn School
of Pathology, South Parks Road, Oxford 0X1 3RE, England.
2
Author's address: Department of Zoology, South Parks Road, Oxford OX1 3PS,
England.
448
C. E. FORD, E. P. EVANS AND R. L. GARDNER
Fig. 1. Chimaera 1.
Fig. 2. Chimaera 2.
of the testis (Mystkowska & Tarkowski, 1968) and in bone marrow, spleen and
thymus (Gornish, Webster & Wegmann, 1972). We have carried out, as an
exploratory investigation, a detailed analysis of two chimaeras obtained by
injection of embryonic cells into recipient blastocysts (Gardner, 1968), both of
which contained cells marked by the T6 chromosome.
MATERIAL AND METHODS
The two chimaeras were obtained by injecting cells from disaggregated 31day post-coitwn (CBA/H-T6 x PDE)/^ blastocysts into the blastocoelic cavity
of synchronous blastocysts of the PDE random-bred albino strain. Chimaera 1
(Fig. 1) was obtained in an experiment in which three cells were injected into
each host blastocyst. and chimaera 2 (Fig. 2) from experiments in which between
two and five cells were injected.
The donor blastocysts were disaggregated in 0-25% trypsin (Difco 1/250)
(Cole & Paul, 1965) following removal of the zona pellucida with 0-5 %pronase
(Mintz, 1962b). Single cells were transferred to a hanging drop in the manipulation chamber via two rinses in medium 199 plus 10% foetal calf serum. Host
Marker chromosome analysis of chimaeras
449
blastocysts were placed in separate individual hanging drops and the disaggregated cells injected into each one with the aid of a Leitz micromanipulator
assembly (Gardner, 1968). Successfully injected blastocysts were transferred to
the uteri of PDE females on the third day of pseudopregnancy after a brief
period in culture.
Fifteen young were born, of which 12 appeared host-type and three were overt
chimaeras. One chimaera and six host-type young died or were killed by the
foster mother post-natally. The remaining eight survived to adulthood and were
paired with PDE mice for test-breeding. All proved to be fertile. Chimaera 1,
a female, produced 28 offspring of which six were pigmented. Chimaera 2, a
male, produced 71 offspring including six pigmented. (Germ cells heterozygous
for the T6 translocation would have produced about 60% chromosomally
unbalanced gametes. Also, half the balanced gametes would have carried the
albino gene. The best estimates of relative output of host-type and donor-type
gametes are therefore 16:30 and 59:30 respectively.) The six surviving host-type
mice yielded only albino offspring. Chimaera 1 was 32 weeks old and chimaera
2, 33 weeks old when killed.
The chimaeras were injected intraperitoneally with 001ml of a 0-04%
(wt/vol) aqueous solution of Colcemid (CIBA), 90 min before they were killed.
Chromosome preparations were made by an air-drying method (Ford, 1966«)
directly (i.e. without intervening in vitro culture) from bone marrow, spleen,
thymus, lymph nodes, and Peyer's patches of both chimaeras. Direct preparations were also obtained from the testes of chimaera 2 by a similar method
(Evans, Breckon & Ford, 1964). Bone marrow specimen 1 was from the left
femur; specimen 2 was from the right femur, tibiae, humeri and iliac bones
combined. Left and right lobes of the thymus from chimaera 1 were processed
independently. Two transverse slices, both about 5 mm thick, were taken from
different parts of the spleen. Eight of the subcutaneous lymph nodes were combined (both axillary, inguinal, deep cervical and superficial cervical nodes); two
more (left and right brachial) were processed separately, as was the mesenteric
node; the three nodes in the lumbar cluster were combined. All Peyer's patches
that could be found (eight in chimaera 1, 11 in chimaera 2) were dissected away
from associated tissue and combined.
Further direct preparations were obtained from corneas and intestinal
epithelium by air-drying from 60 % acetic acid by a method adapted from Fox
& Zeiss (1961). Three segments of the gut were taken, avoiding the sites of
Peyer's patches. Each was about 1 cm long and was divided transversely into
three for processing. PHA-stimulated cultures of whole blood were set up and
harvested on the fifth day, 2 h after injection of Colcemid. Monolayer cultures
were established from multiple small explants of skin, kidney, gut and ovary and
air-dried preparations were obtained from the first sub-culture.
Preparations were scanned systematically at low magnification to select
apparently intact chromosome spreads for identification at high magnification
450
C. E. FORD, E. P. EVANS AND R. L. GARDNER
Table 1. Mitotic cells identified in lymphomyeloid tissues
d.f.: degrees of freedom.
Chimaera 1
Source
Bone marrow 1
Bone marrow 2
Thymus 1
Thymus 2
Peyer's patches
Spleen 1
Spleen 2
LN subcutaneous
LN L. brachial
LN R. brachial
LN mesenteric
LN lumbarBlood culture 1
Blood culture 2
Total
cells
scored
300
200
200
254
200
200
200
300
46
75
200
200
653
181
Chimaera 2
Tests of
homogeneity
%T6
i
cells
9
12
11
11
X2
A
d.f.
^
P
)
1-22 4 - 0 - 9
11 )
28
28
34
24
23
26
718 6 - 0 - 3
28 '
18 1
0-29 1 - 0-7
18 I
Total
cells
scored
200
200
300
—
300
200
200
250
200
200
200
76
581
133
Tests of
homogeneity
%T6
cells
X2
A
d.f.
P
55 )
58
48
_
'2007 3 < 0001
66 /
34
35
32
48
37
41
15-58
6-002
38 '
65
0-54 1 - 0-5
68 J
Significance of differences between bone marrow and spleen groups (percentages transformed to angles): chimaera 1, t = 1610, d.f. = 10, P 0001; chimaera 2, / = 4-89, d.f. =
9, i> 0-001.
by presence or absence of the T6 chromosome. This chromosome is highly
distinctive and we are confident that very few spreads would have been misclassified. There may have been a small bias in favour of normal cells from
occasional artefactual loss of the T6 chromosome as a result of damage during
preparation. However, the eye quickly learns to detect and reject spreads at low
magnification when more than three or four chromosomes are missing and
nearly all spreads arbitrarily chosen for counting contained a full diploid complement of 40 chromosomes. The likelihood of a T6 chromosome being partly
obscured by another chromosome and overlooked is also small. Homogeneity
X2 tests showed that, with two exceptions, data obtained by independent observers
from the same specimen were concordant. Two formally significant #2 values
in 45 tests can be reconciled with random sampling from homogenous populations when the sum of x2 for all tests (xh = 51-50; P ~ 0-5) gives no hint of
heterogeneity. The two sets of apparently discrepant data were therefore accepted.
RESULTS AND DISCUSSION
The results are presented in Tables 1 and 2. The material studied was taken
from 13 different organs or tissues and included representatives of all three
Marker chromosome analysis of chimaeras
451
Table 2. Mitoticjmeiotic cells identified in other tissues and in
monolayer cultures
Chimaera 1
Total
cells
scored
Source
Gut, proximal 1
Gut, proximal 2
Gut, proximal 3
Gut, median 1
Gut, median 2
Gut, median 3
Gut, distal 1
Cornea L
Cornea R
Test is L
Testis R
Skin L*
Skin R*
Kidney L*
Kidney R*
Ovary 1 *
Ovary 2*
Gut*
107
117
96
109
116
101
—
200
271
Chimaera 2
Tests of
homogeneity
%T6
t
X2
cells
d.f.
P
\
35
21
81
Q
16410 10 < 0001
66
43
—
52
•
91 .
z
—
152
200
21
41
117
83
30
52
81
}
32
j
36 1
•
27 j
152
28
90-62
1 < 0-001
• 16-68
1 < 0001
• 13-49
1 < 0001
1-97
Tests of
homogeneity
Total
cells
scored
%T6
100
—
—
200
—
—
100
62
—
—
40
—
—
37
182
76
588f
540J
cells
32 \
58 J
C
A
X2 d.f.
1
P
16-14 2 < 0001
1506 1 < 0001
6 1r 11-92 1 < 0001
26 J
—
278
23
45
29
1 -0-2
* Monolayer cultures.
f 400 spermatogonia (6-5 % T6), 188 spermatocytes (6-4 % T6).
t 400 spermatogonia (25-5 % T6), 140 spermatocytes (25-7 % T6).
embryonic germ layers. Cells derived from both host and donor were identified
in all 53 specimens despite the very small size of some, like single lymph nodes,
corneas and segments of intestine. This result complements the evidence for
fine-grained mixture in morula-aggregation chimaeras provided by investigations such as those of retinal pigmentation (Tarkowski, 1964; Deol & Whitten,
1972), hair structure and pigmentation (Mintz, 1970), and tail rings (McLaren,
Gauld & Bowman, 1973); and also in human chromosome mosaics, for example
the distribution of cells with and without a sex chromatin body in the amnion
and foetal tissues of an XY/XXY abortus (Klinger & Schwarzacher, 1962). The
striking variation in the proportion of donor-type mitoses between corneas and
between separate gut specimens suggests, however, that 'sample sizes' (probably
of the order of 105 proliferating cells) may not have been more than three or
four times greater than the corresponding mean 'patch size'. Whatever the
ultimate patch size the data point to a marked degree of intermingling of cells
29
EM B 33
452
C. E. FORD, E. P. EVANS AND R. L. GARDNER
during embryogenesis, since otherwise large monoclonal blocks and sheets of
tissue would have been expected. To what extent this may be attributed to gross
morphogenetic movements and to what extent to wandering of individual cells
remains an open question.
Blastocysts at the stage chosen for injection consist of some 60 cells, including
a mean of 14-7 cells in the inner cell mass (1CM) (Gardner, 1974). There is at
present no evidence that cells of the trophoblast are capable of contributing to
the embryo (and much to suggest that they do not) (Gardner, 1971). If all 1CM
cells of the recipient blastocysts and all injected cells had contributed equally
to the embryos, the proportion of donor cells would have been 17 % in chimaera
1 and between 17 and 25 % in chimaera 2. But the injected cells would have
included about three trophoblast cells for each ICM cell, so that 'best' estimates
would be 6 % for both chimaeras. The overall mean proportions of donor-type
cells identified were 30 % in chimaera 1 and 40 % in chimaera 2. Despite their
crudeness as measures of total chimaerism the disparity between these figures
is so large as to suggest a much greater than proportional contribution of cells
from the donor in both animals.
A real difference between the relative contributions of host and donor cells
might have come about in several ways. Despite their unnatural position within
the blastocoel the injected cells may have enjoyed an increased likelihood of
being included within the embryo. The low proportion of overt chimaeras in
the series as a whole is not necessarily in conflict since technical factors may
have had an important effect. Differential multiplication or survival of host cells
related to the genotypic difference between donor and host may have played a
part. It is also possible that not all cells, even of the inner cell mass, contribute
to the embryo (Mintz, 1970). Chance exclusion of all the cells of one type could
account for the high frequency of animals with no overt sign of mixed phenotype
in many series of nominal chimaeras produced by morula aggregation (Mullen
& Whitten, 1971); though derived from chimaeric blastocysts, some may no
longer be chimaeras. A similar chance process is implied by the extreme examples
of 'monozygotisme heterocaryote' in which one member of a pair of (human)
monozygotic twins appears to have cells all of one karyotype and the other,
cells all with a different karyotype (Turpin, Lejeune, Lafourcade & Salmon,
1963).
Chimaera 1 had the sex chromosome constitution XXIXX; chimaera 2 was
XYjXY. The young sired by the latter indicated that about 34 % of gametes
were derived from donor-type germ cells whereas direct observations showed
6 and 26 % donor-type spermatocytes in the testes. Selective proliferation or
survival of germ cells in the testes of certain chimaeras has been demonstrated
by Mintz (1968). Though this might account for a difference between young
sired early in life and direct observations on the testis after death, a differential
effect between testes seems unlikely. An alternative explanation would require
the intervention of chance: partition of a small primordial germ cell population
Marker chromosome analysis of chimaeras
453
into two distinct migratory streams, or of equivalent migratory streams (if
indeed there are two) into small effective founder populations in each gonad
and ineffective residua. This could be tested in other chimaeras by estimating
the number of spermatogonial clones per testis, using squash preparations of
single tubules. The great proliferative potential of single germ cells is demonstrated in testicular preparations from mice during recovery after X-irradiation.
Clones of spermatocytes occur of a magnitude that implies repopulation of a
whole tubule by descendants of a single surviving spermatogonium (Ford, 1970).
The lymphomyeloid tissues differ sharply from the remainder in variability
of the proportions of host-type to donor-type mitoses. Whereas there is statistical homogeneity (chimaera 1) or limited heterogeneity (chimaera 2) between
specimens of bone marrow, thymus and Peyer's patches, between spleen and
the different lymph nodes, and between duplicate blood cultures, parallel specimens from tissues or organs not forming part of the lymphomyeloid complex
show marked heterogeneity in seven out of eight tests. These differences imply
a continuing capacity of the proliferating cells of the lymphomyeloid complex
to mix (though apparently in separate pools) that is lacking elsewhere. The
supposition is that in other tissue systems the cellular movements during embryogenesis eventually cease and that continued multiplication leads to the formation
of numerous small local clones.
The differences between the two main groups within the lymphomyeloid
complex are very highly significant in both chimaeras (Table 1). Donor-type
mitoses were more frequent in the spleen group of one animal and in the bone
marrow group of the other. The differences cannot therefore be attributed to a
preference of cells of one genotype for a particular kind of tissue environment.
Investigations of radiation chimaeras (Micklem, Ford, Evans & Gray, 1966),
and spleen colonies (Wu, Till, Siminowitch & McCulloch, 1967) using marker
chromosomes suggested that a single stem cell pool located in bone marrow
supplies the whole lymphomyeloid complex, though evidence was obtained later
for a self-maintaining cell population in lymph nodes (Ford, Micklem & Ogden,
1968). The partition into two groups revealed by the present data was therefore
unexpected. It appears, moreover, to be in direct conflict with data of Gornish
et ah (1972) from morula-aggregation chimaeras, which show statistical homogeneity of proportions of host-type and donor-type mitoses in samples from
bone marrow, spleen and thymus of three out offiveanimals. A possible explanation of this disparity will be offered later.
Earlier investigations using marker chromosomes gave evidence of a flow of
stem cells from bone marrow to thymus (Ford, 1966/?), but no hint of a return
movement or of movement of lymphoid cells into the thymus (Micklem et ah
1966). The discovery in other irradiation experiments (Barnes, Ford, Gray &
Loutit, 1959; C. E. Ford & E. P. Evans, unpublished results) that certain types
of mitotic cells, identified in bone marrow and distinguished by newly induced
unique sets of marker chromosomes, were also present in the thymus and that
29-2
454
C. E. FORD, E. P. EVANS AND R. L. GARDNER
up to 100% of the mitoses sampled could be of a single type, indicated that
some immigrant stem cells were capable of giving rise to enormous clonal
progenies and that the flow might, in fact, be merely a trickle.
The relationships of Peyer's patches with the other tissues of the lymphomyeloid complex had remained enigmatic, although an early result suggested a
partial similarity to lymph nodes in recolonization behaviour (Evans, Ogden,
Ford & Micklem, 1967). The new information places them firmly with bone
marrow and thymus. We assume the deviations from strict homogeneity in
chimaera 2 could be attributed to stochastic fluctuation in the proportions of
host-type and donor-type stem cells moving in relatively small numbers from
bone marrow to both thymus and Peyer's patches, and perhaps partly also to
subsequent unequal clonal amplification.
The close agreement between different bone marrow specimens from the same
animal may be thought unremarkable. Data from parabionts (Ford, 19666)
and from radiation chimaeras (Micklem, Ford, Evans & Ogden, 1975), however,
revealed relatively little exchange of cells between different bone marrow sites
in adult animals. This result could be reconciled with the new data if cell movements in foetal or early post-natal life generated an equilibrium of proportions
in bone marrow that is maintained later, despite greatly diminished exchange
between sites.
It is not surprising that the spleen and the various lymph node specimens
should form a homogeneous group. The rapid and nearly parallel way in which
the mitotic cell populations of spleen and lymph nodes approach equilibrium
proportions in parabionts (Ford, 1966 b) suggests relationship and implies that
the cells concerned are highly mobile. This is concordant with the capacity of
the small lymphocyte to circulate and, when appropriately stimulated, to settle
at some point in lymphatic tissue and transform into a large mononuclear cell
that can re-enter mitosis (Gowans & Knight, 1964). The proliferating cells of
spleen and lymph nodes include progenitors of both B cells and T cells, though
their relative contributions to the mitotic populations in these organs are not
known; the red pulp of the spleen contains myeloid tissue. The close agreement
between spleen and the various lymph nodes from the same animal might then
be explained if B-cell and T-cell precursors normally entered mitosis in approximately the same proportions in different sites, and if the numbers of mitotic
cells in the myeloid tissue of the spleen were too few to influence the proportions
of host-type and donor-type mitoses seriously. The possibility that the ratio
of host-type to donor-type cells in the myeloid tissue of spleen should agree
with that in lymph nodes and differ substantially from bone marrow seems
unlikely, but is open to test by spleen colony analysis.
The mitotic cells in PHA-stimulated blood cultures are thought to be derived
exclusively from the thymus (Davies et al. 1968). Our results are not immediately
reconcilable with this view. However, these cells might be derived from a subpopulation of thymocytes with a distinct ratio of host-type to donor-type cells.
Marker chromosome analysis of chimaeras
455
Alternatively, they may reflect temporal changes in the proportions of different
clones contributing to the circulating thymocyte population.
As already stated, the data given in Table 1 were unexpected and are most
readily interpreted in terms of two independent pools of stem cells, one supplying
bone marrow, thymus and Peyer's patches, the other, the B cells of spleen and
lymph nodes. This interpretation does not necessarily conflict with the wellestablished observation that cells derived from bone marrow can give rise to
functional lymphoid cells in radiation chimaeras: stem cells in bone marrow
might have a reserve capacity to contribute in this way that is rarely called upon
under normal physiological conditions, and the studies of radiation chimaeras
may have given a misleading impression of the magnitude of the normal contribution. Alternatively some stem cells of the lymphoid group might reside in
bone marrow but rarely enter mitosis there.
The possibility that the major lymphocyte populations of spleen and lymph
nodes also consist of small numbers of very large clones each derived from a
single stem cell that originated in a common pool should also be considered.
If this were so, the composition of the mitotic populations in spleen and lymph
nodes jointly, in thymus, and in Peyer's patches would be expected to deviate
independently from the composition of the stem cell pool, the magnitude of the
differences being inversely related to the number of stem cells contributing to
each organ per unit time. Whether limited numbers of randomly originating
clones, distinct for each organ, could have generated the observed within-group
concordances with an acceptable level of probability cannot be answered at
present.
Metcalf & Moore (1971) have recently restated the case for a common origin
of all cells of the lymphomyeloid complex, ultimately in the blood islands of
the yolk sac. This hypothesis could be reconciled with the emergence in the
chimaeras of two functionally differentiated stem cell pools that differed in
relative composition of the two components by again invoking the idea of
stochastic fluctuation arising from low cell numbers. The degree of difference
would be very sensitive to the numbers of founder cells, and if these numbers
should be influenced by the particular genotypes of the components, the separate
populations might be distinguishable by marker chromosome analysis in one
combination but not in another. This may provide an explanation of the apparent
conflict between our results and those reported by Gornish et ah mentioned
earlier, though their sample size was insufficient to detect significant differences
smaller than about 10 % and the disparity may not be a real one.
The foregoing analysis of data from the lymphomyeloid complex has been
founded on four basic assumptions: (1) Embryonic founder populations may
be small enough to permit wide variation in the ratio of host-type and donor-type
cells, though derived from a common pool. (2) Recruitment from the stem cell
pool to the thymus and other organs in post-natal life may be limited. (3) The
magnitude of the clonal progenies produced by individual stem cells may be
456
C. E. FORD, E. P. EVANS AND R. L. GARDNER
variable and sometimes very large. (4) Genotype differences between components may result in differential cellular behaviour, which could be sitedependent and include proliferation, survival, release into the circulation and
removal from it.
These assumptions could be tested, and some of the possible interpretations
discussed excluded, by marker chromosome analysis of further chimaeras in
conjunction with other methods of investigation. Chimaeras of the effectively
isogenic combination CBA/Ca <-> CBA/H-T6 should be particularly informative
and we hope to study them.
We are grateful to Miss S. Harcourt and Mr G. Breckon for setting up the monolayer
cultures and blood cultures respectively and to Miss C. Barnes, Mr M. D. Burtenshaw, Miss
H. Clegg, Miss K. Madan, and Miss J. West, who did much of the scoring. We also wish to
acknowledge valuable advice from Dr A. J. S. Davies, Professor J. L. Gowans, Dr C. F.
Graham, Professor L. J. Lajtha, Dr A. McLaren and Dr H. S. Micklem.
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{Received 26 July 1974, revised 7 October 1974)